Crosslinked chondroitin sulfate, composition containing same, and treatment agent for eye disease

ABSTRACT

Provided is a chondroitin sulfate derivative having a cross-linked structure through a group in a polyvalent amine. Also provided is a composition containing the chondroitin sulfate derivative. Also provided are an agent and method for the treatment of an eye disease, the agent and method having a therapeutic effect on a corneal epithelial disorder and/or dry eye.

This application is a Continuation of U.S. patent application Ser. No.15/543,748, filed Jul. 14, 2017, which is the National Stage ofInternational Patent Application No. PCT/JP2016/051176, filed Jan. 15,2016, which claims the benefit of priority of Japanese Application No.2015-007072, filed Jan. 16, 2015 and Japanese Application No.2015-150976, filed Jul. 30, 2015. The disclosures of each of thesedocuments, including the specifications, drawings and claims, areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a cross-linked chondroitin sulfate, toa composition containing the same, and to an agent for the treatment ofan eye disease.

BACKGROUND ART

According to the international Dry Eye Workshop (DEWS), “dry eye” isdefined as follows: “Dry eye is a multifactorial disease of the tearsand ocular surface that results in symptoms of discomfort, visualdisturbance, and tear film instability with potential damage to theocular surface. It is accompanied by increased osmolarity of the tearfilm and inflammation of the ocular surface” (The Ocular Surface Vol. 5,No. 2: 75-92, 2007).

An eye drop (Chondron (registered trademark)) containing chondroitinsulfate (hereinafter may be referred to as “CS”) has been known inJapan, and the eye drop has the effect of “protecting the superficialcornea.” Studies on the effect of CS on the ocular surface (TsutomuFujihara, et al., Journal of Ocular Pharmacology and Therapeutics, Vol.11, No. 4: 503-508, 1995) have reported the examination of theprevention of corneal epithelial disorders. Teruo Nishida, et al.,Experimental Eye Research, 53: 753-758, 1991 describes the examinationof the presence or absence of the effect of CS on corneal epithelialcells. The recent literature Gary D. Novack, The Ocular Surface, Vol.12, No. 3: 227-230, 2014 describes a variety of therapeutic agents fordry eye developed in Japan and the U.S.

Regarding CS derivatives having a cross-linked structure through across-linker (hereinafter such a derivative may be referred to as“cross-linked CS”) (WO 1991/16881, pamphlet, Japanese Patent ApplicationLaid-Open (kokai) No. 1994-73102, Amnon Sintov, et al., Biomaterials,16: 473-478, 1995), Rubinstein, et al. have reported attempts to produceCS derivatives using 1,12-diaminododecane as a cross-linker (WO1991/16881, pamphlet, Amnon Sintov, et al., Biomaterials, 16: 473-478,1995). In the field of CS, CS (which is readily water-soluble) iscross-linked for imparting poor water-solubility thereto, for use as,for example, membranes or tablets.

PRIOR ART DOCUMENTS Patent Documents

Patent Document 1: WO 1991/16881, pamphlet

Patent Document 2: Japanese Patent Application Laid-Open (kokai) No.1994-73102

Non-Patent Documents

Non-Patent Document 1: The Ocular Surface Vol. 5, No. 2: 75-92, 2007

Non-Patent Document 2: Tsutomu Fujihara, et al., Journal of OcularPharmacology and Therapeutics, Vol. 11, No. 4: 503-508, 1995

Non-Patent Document 3: Teruo Nishida, et al., Experimental Eye Research,53: 753-758, 1991

Non-Patent Document 4: Gary D. Novack, The Ocular Surface, Vol. 12, No.3: 227-230, 2014

Non-Patent Document 5: Amnon Sintov, et al., Biomaterials, 16: 473-478,1995

Non-Patent Document 6: C. Bourie, et al., Journal of BiomaterialsApplications, Vol. 12: 201-221, 1998

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

However, Journal of Ocular Pharmacology and Therapeutics, Vol. 11, No.4: 503-508, 1995 does not disclose the treatment for developed cornealepithelial disorders. Experimental Eye Research, 53: 753-758, 1991describes that CS does not have the effect on migration of cornealepithelial cells. The Ocular Surface, Vol. 12, No. 3: 227-230, 2014 doesnot describe a therapeutic agent containing CS as an activepharmaceutical ingredient.

The cross-linking of CS was not confirmed by various analytical methodsand production methods described in WO 1991/16881, pamphlet orBiomaterials, 16: 473-478, 1995 (C. Bourie, et al., Journal ofBiomaterials Applications, Vol. 12: 201-221, 1998). Therefore, it wasrevealed that no cross-linked CS has been practically produced.

In view of the foregoing, an object of the present invention is toprovide a CS derivative having a cross-linked structure through a groupin a polyvalent amine. Another object of the present invention is toprovide a composition containing the CS derivative. Still another objectof the present invention is to provide an agent for the treatment of aneye disease, the agent having a therapeutic effect on a cornealepithelial disorder and/or dry eye. Yet another object of the presentinvention is to provide a method for the treatment of an eye disease,the method being therapeutically effective on a corneal epithelialdisorder and/or dry eye. The present invention achieves at least one ofthese objects.

Means for Solving the Problems

The present inventors have first successfully produced a CS derivativehaving a cross-linked structure through a group in a polyvalent amineand have accomplished the present invention. The inventors haveunexpectedly found that a composition containing cross-linked CS can beused in the form of a solution, contrary to the conventional knowledgethat CS is cross-linked for imparting poor water solubility thereto. Theinventors have also found that the composition exhibits an excellenttherapeutic effect on a corneal epithelial disorder and/or dry eye. Thepresent invention has been accomplished on the basis of these findings.

The present invention includes the following embodiments.

[A1] A chondroitin sulfate derivative having a cross-linked structurethrough a cross-linker, the derivative being cross-linked betweendisaccharide units of chondroitin sulfate.

[A2] The chondroitin sulfate derivative according to [A1], wherein thecross-linker is a residue derived from at least one species selectedfrom the group consisting of a polyvalent amine, a polyvalent epoxycompound, a polyvalent vinyl compound, and an epihalohydrin.[A3] The chondroitin sulfate derivative according to [A1] or [A2],wherein the cross-linker is a residue derived from a polyvalent amine[A4] The chondroitin sulfate derivative according to [A3], wherein thepolyvalent amine is a polyvalent amine having no biodegradable moiety inthe main chain.[A5] The chondroitin sulfate derivative according to [A3] or [A4],wherein the polyvalent amine is a polyvalent amine having no disulfidebond in the main chain.[A6] The chondroitin sulfate derivative according to any of [A3] to[A5], wherein the polyvalent amine is a substituted or unsubstitutedpolyvalent amine having 1 to 20 carbon atoms in the main chain andoptionally having a heteroatom in the main chain.[A7] The chondroitin sulfate derivative according to any of [A3] to[A5], wherein the polyvalent amine is a substituted or unsubstitutedpolyvalent amine having 1 to 20 atoms in the main chain and optionallyhaving a heteroatom in the main chain.[A8] The chondroitin sulfate derivative according to any of [A3] to[A7], wherein the polyvalent amine is an aliphatic polyvalent amine[A9] The chondroitin sulfate derivative according to any of [A3] to[A8], wherein the polyvalent amine is a diamine.[A10] The chondroitin sulfate derivative according to any of [A3] to[A7], wherein the polyvalent amine is at least one species selected fromthe group consisting of ethane-1,2-diamine, 1,3-diaminopropane,1,4-diaminobutane, 1,5-diaminopentane, 1,6-diaminohexane,1,8-diaminooctane, 1,12-diaminododecane, spermidine, L-lysine ethylester, L-ornithine ethyl ester, 1,3-diamino-2-propanol,2-(aminomethyl)-2-methylpropane-1,3-diamine, (E)-2-butene-1,4-diamine,1,4-bis(aminomethyl)benzene, 1,4-bis(aminomethyl)cyclohexane,2,2′-thiodiethanamine, 2,2′-oxydiethanamine,1,11-diamino-3,6,9-trioxaundecane, and salt forms thereof.[A11] The chondroitin sulfate derivative according to any of [A1] to[A10], wherein the structure is represented by the following formula(I):Y—CO—NH—R—NH—CO—Z  (I)(where Y—CO— represents a disaccharide unit moiety in a chondroitinsulfate molecule;

—CO—Z represents a disaccharide unit moiety in the same chondroitinsulfate molecule or a different chondroitin sulfate molecule;

R represents a substituted or unsubstituted hydrocarbon group optionallyhaving a heteroatom in the main chain; and

—CO—NH— and —NH—CO— each represent an amide bond formed between an aminogroup of a polyvalent amine and a carboxy group of a glucuronic acid,which is a constitutive sugar moiety in chondroitin sulfate).

[A12] The chondroitin sulfate derivative according to [A11], wherein Rin the formula (I) has no biodegradable moiety in the main chain.

[A13] The chondroitin sulfate derivative according to [A11] or [A12],wherein R in the formula (I) has no disulfide bond in the main chain.

[A14] The chondroitin sulfate derivative according to any of [A11] to[A13], wherein R in the formula (I) represents a substituted orunsubstituted hydrocarbon group optionally having a heteroatom in themain chain;

the heteroatom is one to three atoms selected from the group consistingof nitrogen, oxygen, and sulfur; and

the substituted hydrocarbon group has at least one substituent selectedfrom the group consisting of a C1 to C3 alkyl group, a C1 to C3aminoalkyl group, a C1 to C3 hydroxyalkyl group, a C1 to C3 alkyl estergroup, a C1 to C3 alkoxy group, an amino group, a formyl group, ahydroxy group, and a carboxy group.

[A15] The chondroitin sulfate derivative according to any of [A11] to[A14], wherein the hydrocarbon group has 1 to 20 carbon atoms in themain chain.

[A16] The chondroitin sulfate derivative according to any of [A11] to[A14], wherein the hydrocarbon group has 1 to 10 carbon atoms in themain chain.

[A17] The chondroitin sulfate derivative according to any of [A11] to[A14], wherein the hydrocarbon group has 1 to 8 carbon atoms in the mainchain.

[A18] The chondroitin sulfate derivative according to any of [A11] to[A14], wherein the hydrocarbon group has 1 to 20 atoms in the mainchain.

[A19] The chondroitin sulfate derivative according to any of [A11] to[A14], wherein the hydrocarbon group has 1 to 11 atoms in the mainchain.

[A20] The chondroitin sulfate derivative according to any of [A11] to[A14], wherein the hydrocarbon group has 1 to 8 atoms in the main chain.

[A21] The chondroitin sulfate derivative according to any of [A11] to[A20], wherein the hydrocarbon group is an aliphatic hydrocarbon group.

[A22] The chondroitin sulfate derivative according to any of [A1] to[A21], which has water solubility.

[A23] A composition comprising the chondroitin sulfate derivative asrecited in any of [A1] to [A22] and a pharmaceutically acceptablecarrier.

[A24] The composition according to [A23], which is in the form of anaqueous solution.

[A25] The composition according to [A23] or [A24], which passes througha porous filter (pore size: 5.0 μm, diameter: 25 mm) at a passing rateof 80% or more at 25° C.

[A26] The composition according to any of [A23] to [A25], which has aviscosity of 5 to 11,000 mPa·s.

[A27] The composition according to any of [A23] to [A26], wherein theamount of the chondroitin sulfate derivative is 0.1 to 15 wt. % on thebasis of the entire amount of the composition.

[A28] A pharmaceutical preparation comprising the chondroitin sulfatederivative as recited in any of [A1] to [A22].

[A29] The composition according to any of [A23] to [A27], which servesas an agent for the treatment of an eye disease.

[A30] The composition according to any of [A23] to [A27], which servesas an agent for the treatment of a corneal epithelial disorder.

[A31] The composition according to any of [A23] to [A27], which servesas an agent for the treatment of dry eye.

[B1] An agent for the treatment of an eye disease, the agent comprisingthe chondroitin sulfate derivative as recited in any of [A1] to [A22] asan active pharmaceutical ingredient.

[B2] The agent for the treatment of an eye disease according to [B1],which serves as an eye drop.

[B3] The agent for the treatment of an eye disease according to [B1] or[B2], which serves as an aqueous eye drop.

[B4] The agent for the treatment of an eye disease according to any of[B1] to [B3], which is in the form of an aqueous solution.

[B5] The agent for the treatment of an eye disease according to any of[B1] to [B4], which passes through a porous filter (pore size: 5.0 μm,diameter: 25 mm) at a passing rate of 80% or more at 25° C.

[B6] The agent for the treatment of an eye disease according to any of[B1] to [B5], wherein the eye disease is a corneal epithelial disorder.

[B7] The agent for the treatment of an eye disease according to any of[B1] to [B5], wherein the eye disease is dry eye.

[B8] The agent for the treatment of an eye disease according to any of[B1] to [B7], wherein the treatment is therapy.

[C1] A method for the treatment of an eye disease, the method comprisinga step of instilling the composition as recited in any of [A23] to [A27]to an eye of a subject in need thereof.

[C2] The method according to [C1], wherein the composition is instilledinto the eye by instillation.

[C3] The method according to [C1] or [C2], wherein the compositionserves as an aqueous eye drop.

[C4] The method according to any of [C1] to [C3], wherein thecomposition is in the form of an aqueous solution.

[C5] The method according to any of [C1] to [C4], wherein thecomposition passes through a porous filter (pore size: 5.0 μm, diameter:25 mm) at a passing rate of 80% or more at 25° C.

[C6] The method according to any of [C1] to [C5], wherein the eyedisease is a corneal epithelial disorder.

[C7] The method according to any of [C1] to [C5], wherein the eyedisease is dry eye.

[C8] The method according to any of [C1] to [C7], wherein the treatmentis therapy.

Advantageous Effects of the Invention

In one aspect, the present invention provides a CS derivative having across-linked structure through a group in a polyvalent amine. In anotheraspect, the present invention provides a composition containing thecross-linked CS derivative (hereinafter referred to as “cross-linkedCS”). In still another aspect, the present invention provides an agentfor the treatment of an eye disease, the agent having a therapeuticeffect on a corneal epithelial disorder and/or dry eye. In yet anotheraspect, the present invention provides a method for the treatment of aneye disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows HPLC charts of chondroitinase-ABC-digested liquids.

FIG. 2 shows an example of the results of analysis by multi-stage massspectrometry.

FIG. 3 shows an example of the results of analysis by multi-stage massspectrometry.

FIG. 4 shows a graph illustrating an average degree of fluoresceinstaining in the case of ocular instillation into an animal model twice aday.

FIG. 5 shows a graph illustrating an average degree of fluoresceinstaining in the case of ocular instillation into an animal model twice aday.

FIG. 6 shows a graph illustrating an average degree of fluoresceinstaining in the case of ocular instillation into an animal model twice aday.

FIG. 7 shows a graph illustrating an average degree of fluoresceinstaining in the case of ocular instillation into an animal model twice aday.

FIG. 8 shows a graph illustrating an average degree of fluoresceinstaining in the case of ocular instillation into an animal model twice aday.

FIG. 9 shows a graph illustrating an average degree of fluoresceinstaining in the case of ocular instillation into an animal model twice aday.

FIG. 10 shows a graph illustrating an average degree of fluoresceinstaining in the case of ocular instillation into an animal model twice aday.

MODES FOR CARRYING OUT THE INVENTION

Terms as used herein will now be defined as follows. The term“chondroitin sulfate” (may be referred to as “CS”) refers to chondroitinsulfate or a salt form thereof. The term “cross-linked chondroitinsulfate” (may be referred to as “cross-linked CS”) refers to a CSderivative or a salt form thereof having a cross-linked structurethrough a cross-linker, the derivative being cross-linked betweendisaccharide units of CS. The derivative may be cross-linked within asingle CS molecule and/or between different CS molecules.

As used herein, the term “substitution” refers to substitution of one ormore hydrogen atoms in a compound by another atom or a group of atoms.The atom or group of atoms substituted for the hydrogen atom(s) will bereferred to as “substituent.” The term “heteroatom” refers to an atom(other than carbon and hydrogen) contained in a compound, such asnitrogen, oxygen, or sulfur. Unless otherwise specified, the expression“having a heteroatom” refers to the case where a compound has one ormore types of heteroatoms, and one or more atoms of each type arepresent in the compound. The expression “having a heteroatom in a chain”refers to the case where at least one carbon atom in the chain issubstituted by a heteroatom. The term “carbon chain” refers to an atomicchain mainly formed of carbon atoms, the chain optionally having abranch and/or a heteroatom. The term “reactive functional group” refersto a functional group having reactivity with a functional groupcontained in chondroitin sulfate. Unless otherwise specified, the term“main chain” refers to a linear carbon chain which links two reactivefunctional groups. When three or more reactive functional groups arepresent, the main chain is the longest one of linear carbon chains eachlinking two reactive functional groups. When the carbon chain has abranch, unless otherwise specified, a carbon chain of a branch is notincluded in the main chain. The main chain may have one or moreheteroatoms at any position of a carbon atom other than a position atwhich the carbon atom is directly bonded to a reactive functional group.When a cyclic structure is present in a carbon chain linking reactivefunctional groups, the cyclic structure is regarded as having at leastone branch and sharing an atom as a terminal of at least two branchedcarbon chains. In this case, the main chain is the longest chain amongeach of the branched chains. As used herein, the “number of atoms in amain chain” refers to the number of atoms constituting the main chain.In the case where the main chain has a heteroatom, the number of atomsin the main chain includes the number of the heteroatom. As used herein,the “main chain” of a polyvalent amine or a group in a polyvalent aminerefers to a linear carbon chain linking two primary amino groups, whichare reactive functional groups.

As used herein, the term “hydrocarbon” refers to, for example, a groupor compound having carbon and hydrogen. Unless otherwise specified, thehydrocarbon may have a heteroatom and/or a substituent. The“hydrocarbon” may have a linear or branched structure. The term“aliphatic” refers to, for example, a group or compound having noaromaticity. The term “cyclic” refers to, for example, a group orcompound having an intramolecular cyclic structure. The term “acyclic”refers to, for example, a group or compound having no intramolecularcyclic structure. The term “alkyl group” refers to a monovalent linearor branched saturated aliphatic hydrocarbon group. The term “alkylenegroup” refers to a divalent linear or branched saturated aliphatichydrocarbon group. The term “alkenylene group” refers to a divalentlinear or branched unsaturated aliphatic hydrocarbon group having atleast one double bond. The term “alkynylene group” refers to a divalentlinear or branched unsaturated aliphatic hydrocarbon group having atleast one triple bond. The term “aryl group” refers to a monovalentmonocyclic or polycyclic aromatic hydrocarbon group. The term “arylenegroup” refers to a divalent monocyclic or polycyclic aromatichydrocarbon group. The term “arylalkylene group” refers to a divalentgroup formed through substitution of one hydrogen atom of an aryl groupby an alkylene group, or a divalent group formed through substitution oftwo hydrogen atoms of an aromatic ring by alkylene groups. The term“cycloalkyl group” refers to a monovalent cyclic aliphatic hydrocarbongroup. The term “cycloalkylene group” refers to a divalent cyclicaliphatic hydrocarbon group. The term “cycloalkylalkylene group” refersto a divalent group formed through substitution of one hydrogen atom ofa cycloalkyl group by an alkylene group, or a divalent group formedthrough substitution of two hydrogen atoms of a cycloalkane by alkylenegroups. The term “aminoalkyl group” refers to an amino-substituted alkylgroup. The term “hydroxyalkyl group” refers to a hydroxy-substitutedalkyl group.

As used herein, the term “polyvalent amine” refers to an amine havingtwo or more primary amino groups (—NH₂). As used herein, the term“diamine” refers to an amine having two primary amino groups, and theterm “triamine” refers to an amine having three primary amino groups.

As used herein, the term “aliphatic polyvalent amine” refers to apolyvalent amine formed through substitution of two or more hydrogenatoms of an aliphatic hydrocarbon by primary amino groups. The term“aromatic polyvalent amine” refers to a polyvalent amine formed throughsubstitution of two or more hydrogen atoms of an aromatic hydrocarbon byprimary amino groups or aminoalkyl groups.

As used herein, the term “to” between two numerical values indicatesthat the numerical values before and after the term mean the lower limitvalue and the upper limit value in the range, respectively. When acomposition contains some kind of components, the content of thecomponent in the composition refers to the total amount of thecomponents in the composition, unless otherwise specified.

The present invention will next be described in more detail withreference to embodiments, which should not be construed as limiting theinvention thereto.

<Cross-Linked CS>

The “cross-linked CS” of the present invention has a cross-linkedstructure between disaccharide units of CS through a cross-linker. Thecross-linked CS may be cross-linked within a single CS molecule and/orbetween different CS molecules. No particular limitation is imposed onthe type of CS used as a raw material for producing the cross-linked CS,so long as the CS is a glycosaminoglycan that has a primary structureformed of repeating disaccharide units of D-glucuronic acid andN-acetyl-D-galactosamine wherein hydroxy groups of the constitutivesugars are partially sulfated. The cross-linked CS and CS used as a rawmaterial therefor may be in a free (non-salt) form or a pharmaceuticallyacceptable salt form.

Examples of the pharmaceutically acceptable salt include an alkalimetal, such as a sodium salt and a potassium salt; and an alkaline earthmetal salt, such as a magnesium salt and a calcium salt. Thecross-linked CS of the present invention is preferably in the form of apharmaceutically acceptable alkali metal salt, more preferably in theform of a sodium salt, from the viewpoints of biological compatibilityand affinity.

The cross-linked CS of the present invention or CS used as a rawmaterial therefor may be in the form of a hydrate or a solvate.

No particular limitation is imposed on the type of CS used as a rawmaterial for producing the cross-linked CS, and the CS may be, forexample, chondroitin sulfate C (hereinafter may be referred to as“CSC”). No particular limitation is imposed on the origin of CS used asa raw material for producing the cross-linked CS. The CS may be anaturally occurring substance or may be a chemically synthesizedsubstance. In the case where CS is obtained from, for example, anaturally occurring product, the naturally occurring product (i.e., rawmaterial) may be appropriately selected depending on the type of CS ofinterest and so on. Alternatively, CS of interest may be preparedthrough appropriate modification of a naturally occurring substance by achemosynthetic technique.

No particular limitation is imposed on the weight average molecularweight of CS used as a raw material for producing the cross-linked CS.The weight average molecular weight of CS is preferably 10,000 to100,000, more preferably 10,000 to 80,000, still more preferably 10,000to 60,000, particularly preferably 15,000 to 45,000. The weight averagemolecular weight of CS can be determined by a common technique, such assize exclusion chromatography or a light scattering method.

The cross-linked CS can be produced through covalent bonding between CSand a cross-linking agent.

<Cross-Linker>

As used herein, the term “cross-linker” refers to a residue derived froma compound having two or more groups capable of covalently bonding tothe groups contained in CS. Such a compound preferably has at least onegroup selected from the group consisting of an amino group, an epoxygroup, a vinyl group, and a haloalkyl group, wherein the number of theselected group(s) is two or more. The cross-linked CS of the presentinvention is preferably a cross-linked form with such a group. Thecompound having two or more groups capable of covalently bonding to thegroups contained in CS is more preferably at least one compound selectedfrom the group consisting of polyvalent amines, polyvalent epoxycompounds, polyvalent vinyl compounds, and epihalohydrins.

No particular limitation is imposed on the number of carbon atoms of thecross-linker used in the present invention or the number of carbon atomsof the polyvalent amine, polyvalent epoxy compound, polyvalent vinylcompound, and epihalohydrin used in the present invention. The number ofthe carbon atoms is, for example, 1 to 20, 1 to 12, 1 to 10, 1 to 8, 1to 6, 1 to 4, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 12, 4 to10, or 4 to 8.

The amino group can form an amide bond with the carboxy group of CS. Theepoxy group, the vinyl group, or the haloalkyl group can form an etherbond with a hydroxy group of CS. CS has only one carboxy group in thedisaccharide unit moiety. Thus, the cross-linker preferably has an aminegroup, and is particularly preferably a polyvalent amine, in order toreduce the structural diversity of the cross-linked CS. No particularlimitation is imposed on the polyvalent amine, so long as it has two ormore primary amino groups (—NH₂). The polyvalent amine may be, forexample, a triamine or a diamine. The polyvalent amine is preferably adiamine for reducing the structural diversity of the cross-linked CS.

In the present invention, the polyvalent amine may be a polyvalent aminehaving no biodegradable bond (i.e., no biodegradable moiety). In thepresent invention, the polyvalent amine may be a polyvalent amine havingno biodegradable moiety in the main chain of the molecule. As usedherein, the term “biodegradable moiety” refers to a structurerepresented by the following formula (II):—C(═O)—D—  (II)(where D represents an oxygen atom or a sulfur atom). Specifically, thebiodegradable moiety corresponds to an ester bond or a thioester bond.Thus, in the present invention, the polyvalent amine may be a polyvalentamine having neither an ester bond nor a thioester bond in the mainchain of the molecule.

In the present invention, the polyvalent amine may be a polyvalent aminehaving no disulfide bond. The polyvalent amine may be a polyvalent aminehaving no disulfide bond in the main chain.

In the present invention, the polyvalent amine may be, for example, analiphatic polyvalent amine or an aromatic polyvalent amine, but ispreferably an aliphatic polyvalent amine. The aliphatic polyvalent aminemay be an acyclic aliphatic polyvalent amine or a cyclic aliphaticpolyvalent amine, but is preferably an acyclic aliphatic polyvalentamine. The aliphatic polyvalent amine may be a saturated aliphaticpolyvalent amine or an unsaturated aliphatic polyvalent amine That is,the aliphatic polyvalent amine may be an acyclic saturated aliphaticpolyvalent amine, an acyclic unsaturated aliphatic polyvalent amine, acyclic saturated aliphatic polyvalent amine, or a cyclic unsaturatedaliphatic polyvalent amine, but is preferably an acyclic saturatedaliphatic polyvalent amine. The polyvalent amine may be the same as abiological component or a derivative thereof. For example, thepolyvalent amine may be a basic amino acid or a derivative thereof.

In the present invention, the polyvalent amine may be, for example, asubstituted or unsubstituted, aliphatic polyvalent amine or aromaticpolyvalent amine optionally having a heteroatom in the main chain.

In the present invention, examples of the substituent include an alkylgroup, an aminoalkyl group, a hydroxyalkyl group, an alkyl ester group,an alkoxy group, an amino group, a formyl group, a hydroxy group, acarboxy group, and a carbonyl group. When the substituent contains areactive functional group (e.g., an aminoalkyl group), the substituentdoes not form the main chain of the polyvalent amine. The substituent ispreferably an alkyl group, an aminoalkyl group, an alkyl ester group, ora hydroxy group. The alkyl group, the aminoalkyl group, the hydroxyalkylgroup, the alkyl ester group, and the alkoxy group each preferably haveone to five carbon atoms, more preferably one to three carbon atoms. Thealkyl group is preferably a C1 to C3 alkyl group; i.e., a methyl group,an ethyl group, or a propyl group. The aminoalkyl group is preferably anaminomethyl group, an aminoethyl group, or an aminopropyl group.Examples of the alkyl ester group include a methyl ester group, an ethylester group, a propyl ester group, and a butyl ester group. Of these,preferred is a methyl ester group or an ethyl ester group. Thepolyvalent amine may have any number of substituents. The polyvalentamine may have one to five substituents at substitutable positions. Thepolyvalent amine preferably has one to three substituents. If thepolyvalent amine has two or more substituents, the substituents may beidentical to or different from one another.

In the present invention, examples of the heteroatom include nitrogen,oxygen, and sulfur. The polyvalent amine may have any number ofheteroatoms in the main chain. The polyvalent amine has, for example,one to five heteroatoms or one to three heteroatoms. When the polyvalentamine has two or more heteroatoms, the heteroatoms may be identical toor different from one another. When the polyvalent amine has two or moreheteroatoms in the main chain, these heteroatoms may be locatedseparately so as to prevent direct bonding therebetween. When thepolyvalent amine has a heteroatom in the main chain, the heteroatom maybe located at a position other than a position where a reactivefunctional group (in particular, a primary amino group) is directlybonded.

In the present invention, the number of carbon atoms in the main chainof the polyvalent amine is, for example, 1 or more, or 2 or more, andis, for example, 20 or less, 12 or less, 10 or less, 8 or less, 6 orless, or 4 or less. The number of carbon atoms is, for example, 1 to 20,1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 2 to 20, 2 to 12, 2 to 10, 2to 8, 2 to 6, 2 to 4, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Thenumber of carbon atoms in the main chain of the polyvalent amine ispreferably 1 to 10, more preferably 1 to 8, still more preferably 1 to6, particularly preferably 2 to 4, from a viewpoint of an improvement incross-linking efficiency. The number of carbon atoms in the main chainof the polyvalent amine is preferably 1 to 10, more preferably 1 to 8,still more preferably 1 to 6, yet more preferably 2 to 5, yet still morepreferably 2 to 4, from a viewpoint of an improvement in medicalefficacy.

In the present invention, the number of atoms in the main chain of thepolyvalent amine is, for example, 1 or more, or 2 or more, and is, forexample, 20 or less, 12 or less, 11 or less, 10 or less, 8 or less, 6 orless, or 4 or less. The number of atoms is, for example, 1 to 20, 1 to12, 1 to 11, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 2 to 20, 2 to 12, 2 to 11,2 to 10, 2 to 8, 2 to 6, 2 to 4, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or12. The number of atoms in the main chain of the polyvalent amine ispreferably 1 to 11, more preferably 1 to 10, still more preferably 1 to8, yet more preferably 1 to 6, particularly preferably 2 to 4, from aviewpoint of an improvement in cross-linking efficiency. The number ofatoms in the main chain of the polyvalent amine is preferably 1 to 11,more preferably 1 to 10, still more preferably 1 to 8, yet morepreferably 1 to 6, yet still more preferably 2 to 5, particularlypreferably 2 to 4, from a viewpoint of an improvement in medicalefficacy.

In the present invention, the polyvalent amine is preferably a triamineor a diamine, and more preferably a diamine.

In the present invention, the polyvalent amine is preferably analiphatic diamine or an aromatic diamine, and more preferably analiphatic diamine. The aliphatic diamine may be, for example, an acyclicaliphatic diamine or a cyclic aliphatic diamine, but is preferably anacyclic aliphatic diamine. Examples of the cyclic aliphatic diamineinclude a cycloalkylenediamine and a bis(aminoalkyl)cycloalkane.Examples of the acyclic aliphatic diamine include an alkylenediamine, analkenylenediamine, and an alkynylenediamine. Of these, analkylenediamine and an alkenylenediamine are preferred, and analkylenediamine is particularly preferred. Examples of the aromaticdiamine include an arylenediamine and a bis(aminoalkyl)benzene.

In the present invention, specific examples of the polyvalent amineinclude an alkylenediamine, such as ethane-1,2-diamine,1,3-diaminopropane, 1,4-diaminobutane (putrescine), 1,5-diaminopentane(cadaverine), 1,6-diaminohexane, 1,7-diaminoheptane, 1,8-diaminooctane,1,9-diaminononane, 1,10-diaminodecane, 1,11-diaminoundecane,1,12-diaminododecane, 1,3-diamino-2-propanol, 2,2′-thiodiethanamine,2,2′-oxydiethanamine, 1,11-diamino-3,6,9-trioxaundecane, L-lysine ethylester, L-ornithine ethyl ester, N-(2-aminoethyl)-1,2-ethanediamine,N-(3-aminopropyl)butane-1,4-diamine (spermidine), andN,N′-bis(3-aminopropyl)butane-1,4-diamine (spermine); triamines, such aspropane-1,2,3-triamine, pentane-1,3,5-triamine, and2-(aminomethyl)-2-methylpropane-1,3-diamine;1,4-bis(aminomethyl)cyclohexane; 1,4-bis(aminomethyl)benzene;(E)-2-butene-1,4-diamine; and a salt form thereof. These amines may beused singly or in combination of two or more species.

In the present invention, no particular limitation is imposed on thepolyvalent epoxy compound, so long as it has two or more epoxy groups.Examples of the diepoxy compound include a diglycidyl compound.

In the present invention, no particular limitation is imposed on thepolyvalent vinyl compound, so long as it has two or more vinyl groups.Examples of the divinyl compound include a divinyl sulfone.

In the present invention, examples of the epihalohydrin include anepichlorohydrin.

<Cross-Linked Structure>

As used herein, the term “cross-linked structure” refers to a structureformed within a single CS molecule or between different CS moleculesthrough covalent bonding with a cross-linker.

When the cross-linker is a polyvalent amine, the cross-linked CS of thepresent invention may have a cross-linked structure represented by thefollowing formula (I):Y—CO—NH—R—NH—CO—Z  (I)(where Y—CO— represents a disaccharide unit moiety in a chondroitinsulfate molecule;

—CO—Z represents a disaccharide unit moiety in the same chondroitinsulfate molecule or a different chondroitin sulfate molecule;

R represents a substituted or unsubstituted hydrocarbon group optionallyhaving a heteroatom in the main chain; and

—CO—NH— and —NH—CO— each represent an amide bond formed between an aminogroup of a polyvalent amine and a carboxy group of a glucuronic acid,which is a constitutive sugar moiety in chondroitin sulfate).

When the cross-linker is a polyvalent amine, the cross-linked CS of thepresent invention may have a structure represented by the followingformula (III):Y—CO—NH—R—NH₂  (III)(where Y—CO—, R, and —CO—NH— each represent the same embodiments asdefined in the formula (I) above). That is, the cross-linked CS of thepresent invention may have a structure in which one amino group of thepolyvalent amine is covalently bonded to CS.

In the formula (I) or (III), —NH—R—NH— or —NH—R—NH₂ is a group in apolyvalent amine. Thus, the description, examples, and preferred rangesregarding the polyvalent amine in the present invention described abovein the section <cross-linker> can be applied mutatis mutandis to those.

R in the formulae (I) and (III) may be a substituted or unsubstitutedhydrocarbon group optionally having a heteroatom in the main chain. Thehydrocarbon group may be an aliphatic hydrocarbon group or an aromatichydrocarbon group, but is preferably an aliphatic hydrocarbon group. Thealiphatic hydrocarbon group may be an acyclic aliphatic hydrocarbongroup or a cyclic aliphatic hydrocarbon group, but is preferably anacyclic aliphatic hydrocarbon group. The aliphatic hydrocarbon group maybe a saturated aliphatic hydrocarbon group or an unsaturated aliphatichydrocarbon group. That is, the aliphatic hydrocarbon group may be anacyclic saturated aliphatic hydrocarbon group, an acyclic unsaturatedaliphatic hydrocarbon group, a cyclic saturated aliphatic hydrocarbongroup, or a cyclic unsaturated aliphatic hydrocarbon group, but ispreferably an acyclic saturated aliphatic hydrocarbon group. Examples ofthe acyclic aliphatic hydrocarbon group include an alkylene group, analkenylene group, and an alkynylene group. Of these, an alkylene groupor an alkenylene group is preferred, and an alkylene group is morepreferred. Examples of the cyclic aliphatic hydrocarbon group include acycloalkylene group and a cycloalkylalkylene group. Examples of thearomatic hydrocarbon group include an arylene group and an arylalkylenegroup. R in the formulae (I) and (III) may be a hydrocarbon group otherthan an unsubstituted hydrocarbon group having no heteroatom in the mainchain.

The description, examples, and preferred ranges regarding the polyvalentamine in the present invention described above in the section<cross-linker> can be applied mutatis mutandis to the substituent andheteroatom in the present invention.

R in the formulae (I) and (III) may be a hydrocarbon group having nobiodegradable moiety. Alternatively, R may be a hydrocarbon group havingno biodegradable moiety in the main chain. The biodegradable moiety maybe, for example, an ester bond or a thioester bond. Thus, R may be ahydrocarbon group having neither an ester bond nor a thioester bond inthe main chain.

R in the formulae (I) and (III) may be a hydrocarbon group having nodisulfide bond. Alternatively, R may be a hydrocarbon group having nodisulfide bond in the main chain. When R has two or more heteroatoms inthe main chain, these heteroatoms may be located so as to prevent directbonding therebetween. In the formulae (I) and (III), the terminal atomsof R bonded to the nitrogen atoms may be carbon atoms.

The description, examples, and preferred ranges regarding the number ofcarbon atoms and atoms in the main chain of the polyvalent amine in thepresent invention described above in the section <cross-linker> can beapplied mutatis mutandis to the number of carbon atoms and atoms in themain chain of R in the formulae (I) and (III).

In one embodiment of the cross-linked CS of the present invention, R inthe formulae (I) and (III) is —CH₂CH₂—(R¹—CH₂CH₂)_(n)— (where R¹represents an oxygen atom, a sulfur atom, or —NH; when n is 2 or more,the radicals R¹ may be identical to or different from one another; and nrepresents an integer of 1 to 5 or 1 to 3).

In one embodiment of the cross-linked CS of the present invention, R inthe formulae (I) and (III) is —(CH₂)₁—(CR²R³)—(CH₂)_(m)— (where R² andR³ each independently represent a hydrogen atom, —OH, —NH₂, or a C1 toC3 alkyl group, alkyl ester group, or aminoalkyl group; 1 represents aninteger of 1 to 5; and m represents an integer of 0 to 5). In a furtherembodiment, R is —(CH₂)₁—(CR²R³)—(CH₂)_(m)— (where R², R³, 1, and m arethe same as defined above; and when R² is hydrogen atom, R³ is not ahydrogen atom). In another further embodiment, R is—(CH₂)₁—(CR²R³)—(CH₂)_(m)— (where R², R³, and 1 are the same as definedabove; m represents an integer of 1 to 5; and when R² is hydrogen atom,R³ is not a hydrogen atom).

In one embodiment of the cross-linked CS of the present invention, R inthe formulae (I) and (III) is —(CH₂)_(p)—CH═CH—(CH₂)_(q)— (where p and qeach independently represent an integer of 0 to 3 or 1 to 2).

Specific examples of R in the formulae (I) and (III) include —(CH₂)₂—,—(CH₂)₃—, —(CH₂)₄—, —(CH₂)₅—, —(CH₂)₆—, —(CH₂)₇—, —(CH₂)₃—, —(CH₂)₉—,—(CH₂)₁₀—, —(CH₂)₁₁—, —(CH₂)₁₂—, —CH₂CH═CHCH₂—, —CH₂CH(OH)CH₂—,—CH₂CH₂—S—CH₂CH₂—, —CH₂CH₂—O—CH₂CH₂—, —CH₂CH₂O—CH₂CH₂—CH₂CH₂—CH₂CH₂—,CH₂CH(NH₂)CH₂—, —CH₂CH₂CH(NH₂)CH₂CH₂—, —CH₂CH₂—NH—CH₂CH₂—, —CH₂—X—CH₂—,—CH₂-Ph-CH₂—, —CH₂C(CH₂NH₂)(CH₃)CH₂—, —(CH₂)₄—NH—(CH₂)₃—,—(CH₂)₃—NH—(CH₂)₄—NH—(CH₂)₃—, —(CH₂)₄—CH(COOCH₂CH₃)—, and—(CH₂)₃—CH(COOCH₂CH₃)— (where X represents a 1,4-cyclohexylene group,and Ph represents a 1,4-phenylene group).

If the cross-linker is a polyvalent amine, all the carboxy groups in CSdo not necessarily form amide bonds with the cross-linker; i.e., some ofthe carboxy groups may form amide bonds with the cross-linker.

One embodiment of the cross-linked CS of the present invention is asfollows.

A CS derivative having a cross-linked structure formed with a group in apolyvalent amine, the cross-linked structure being present betweendisaccharide units of CS, wherein the cross-linked structure isrepresented by the following formula (I):Y—CO—NH—R—NH—CO—Z  (I)(where Y—CO— represents a disaccharide unit moiety in a CS molecule;

—CO—Z represents a disaccharide unit moiety in the same CS molecule or adifferent CS molecule;

R represents a substituted or unsubstituted aliphatic hydrocarbon grouphaving 1 to 11 atoms in the main chain and optionally having aheteroatom in the main chain, the heteroatom is one to three atomsselected from the group consisting of nitrogen, oxygen, and sulfur, andthe substituted hydrocarbon group has at least one substituent selectedfrom the group consisting of a C1 to C3 alkyl group, a C1 to C3aminoalkyl group, a C1 to C3 hydroxyalkyl group, a C1 to C3 alkyl estergroup, a C1 to C3 alkoxy group, an amino group, a formyl group, ahydroxy group, and a carboxy group; and

—CO—NH— and —NH—CO— each represent an amide bond formed between an aminogroup of a polyvalent amine and the carboxy group of glucuronic acid,which is a constitutive sugar of CS). In the cross-linked CS accordingto this embodiment, R in the formula (I) may be a hydrocarbon group thatdoes not have a biodegradable moiety and/or a disulfide bond in the mainchain.

<Method for Producing Cross-Linked CS>

The cross-linked CS of the present invention can be produced throughcovalent bonding of two functional groups of CS and two reactivefunctional groups of a cross-linking agent by a common process. The twofunctional groups of CS may be present in a single CS molecule or indifferent CS molecules. The cross-linking agent may be a polyfunctionalcross-linking agent, such as a polyvalent amine, a polyvalent epoxycompound, a polyvalent vinyl compound, or an epihalohydrin. The examplesand preferred ranges described above in the section <cross-linker> canbe applied mutatis mutandis to the linking agent.

The concentration of CS in a solvent used for the cross-linking reactionbetween CS and the cross-linking agent is preferably 2 to 20% by weight(hereinafter may be referred to as “wt. %”), more preferably 2 to 15 wt.%, particularly preferably 3 to 15 wt. %.

No particular limitation is imposed on the solvent used forcross-linking reaction, so long as it dissolves CS and the cross-linkingagent. The solvent is preferably water or a mixture of water and awater-miscible organic solvent. Examples of the water-miscible organicsolvent include, but are not particularly limited to, lower alcohols,such as methanol, ethanol, isopropanol, n-propanol, and tertiarybutanol; glycol ethers, such as ethylene glycol monomethyl ether andethylene glycol monoethyl ether; acetone; 1,4-dioxane; tetrahydrofuran;and acetonitrile. Of these, methanol, ethanol, acetone, tetrahydrofuran,and 1,4-dioxane are preferred. These water-miscible organic solvents,which are mixed with water, may be used singly or in combination of twoor more species.

No particular limitation is imposed on the reaction time ofcross-linking. The reaction time of cross-linking may be, for example, 1to 48 hours, 1 to 24 hours, 2 to 20 hours, or overnight. As used herein,the term “overnight” refers to 10 to 24 hours. The cross-linkingreaction may be stopped during the reaction by, for example, addition of10% aqueous sodium carbonate solution. No particular limitation isimposed on the reaction temperature of cross-linking. The reactiontemperature of cross-linking, which is appropriately determineddepending on the type of the solvent used, is preferably 5 to 60° C.,more preferably 15 to 30° C.

The cross-linking reaction may be appropriately followed by an alkalitreatment step. No particular limitation is imposed on the alkalitreatment for alkalifying the reaction mixture obtained through thecross-linking reaction, so long as the treated reaction mixture exhibitsalkalinity. Specifically, the alkali treatment preferably involves theuse of an inorganic base. In particular, sodium hydroxide, sodiumhydrogen carbonate, or sodium carbonate is preferably used. The alkalitreatment is performed at a pH of, for example, 7.2 to 11, preferably ata pH of 7.5 to 10. No particular limitation is imposed on the time ofalkali treatment. The time of alkali treatment is, for example, 2 to 12hours, preferably 2 to 6 hours. The temperature of alkali treatment ispreferably 5 to 60° C., more preferably 15 to 30° C.

After the cross-linking reaction or the alkali treatment step, thereaction mixture containing cross-linked CS is subjected to 1) anagitation step, 2) a precipitation step, 3) a washing step, and 4) adrying step, thereby finally preparing a dry powder of cross-linked CS.

The agitation step, the precipitation step, the washing step, and thedrying step may be carried out by a method generally known to thoseskilled in the art. No particular limitation is imposed on the method.

<Cross-Linking of CS Using Polyvalent Amine as Cross-Linking Agent>

When a polyvalent amine is used as a cross-linking agent, CS can becross-linked through covalent bonding between a carboxy group of CS andan amino group of the cross-linking agent by a common amidation method.

In this case, the amidation method may be, for example a method using,in a solvent, a condensing agent, such as a water-soluble carbodiimide(e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide),dicyclohexylcarbodiimide (DCC), or4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride Nhydrate (DMT-MM); the symmetric acid anhydride method; the mixed acidanhydride method; or the active ester method. The reaction conditionsfor the amidation method are appropriately determined depending on theCS and cross-linking agent used.

In the case of the cross-linking reaction between a carboxy group of CSand a polyvalent amine as a cross-linking agent, the concentration of CSin a solvent is preferably 2 to 20 wt. %, more preferably 2 to 15 wt. %,particularly preferably 3 to 15 wt. %.

The mole equivalent (eq) of the polyvalent amine as a cross-linkingagent is preferably 0.005 to 0.500 eq, more preferably 0.005 to 0.300eq, still more preferably 0.005 to 0.250 eq, yet more preferably 0.005to 0.200 eq, particularly preferably 0.005 to 0.100 eq, relative to 1.00mole equivalent (eq) of the disaccharide unit of CS.

The mole equivalent (eq) of the condensing agent is preferably [(0.50 to5.00)×valence] eq, more preferably [(1.00 to 4.00)×valence] eq,particularly preferably [(1.00 to 3.00)×valence] eq, relative to 1.00mole equivalent (eq) of the polyvalent amine as a cross-linking agent.As used herein, the term “valence” refers to the valence of thepolyvalent amine.

The examples and preferred ranges described above can be applied mutatismutandis to the solvent used for the cross-linking reaction, thereaction time of cross-linking, and the reaction temperature ofcross-linking. The amidation method used for the cross-linking reaction,the CS concentration, the structure of the polyvalent amine as across-linking agent, the amount of the cross-linking agent added, theamount of the condensing agent added, and the solvent are appropriatelydetermined depending on the properties required for the cross-linked CS.

In the case of cross-linking by use of a hydroxy group of CS,cross-linked CS products having a variety of structures may be produceddepending on the cross-linked position of the hydroxy groups. Thus, thecross-linking by use of a carboxy group of CS is preferably selected forreducing the structural diversity of the cross-linked CS. Thecross-linking agent is preferably a polyvalent amine in view offormation of an amide bond between the polyvalent amine and a carboxygroup of CS. In order to reduce the structural diversity of thecross-linked CS, a diamine is particularly preferred.

The cross-linked structure of the cross-linked CS can be determined by,for example, the method described in Example 12A.

<Percent of Cross-Linking>

As used herein, the percent of cross-linking of the cross-linked CSrefers to the percentage of the number of cross-linkers (wherein two ormore functional groups are bonded to CS) relative to the number ofdisaccharide units of CS. For example, the percent of cross-linking of aCS derivative having a cross-linked structure formed with a group in adiamine is calculated by the following formula (A):

[Formula 1](the amount by mole (mol) of a cross-linker wherein both terminal aminogroups are bonded to CS)/(the total amount by mole (mol) of —COR in thecross-linked CS)×100(%)  (A)(where R represents —OH, —OM (M represents a metal of group 1 or 2 ofthe periodic table), or a group in a diamine).

No particular limitation is imposed on the percent of cross-linking ofthe cross-linked CS of the present invention. The percent ofcross-linking may be, for example, 0.01% or more, 0.05% or more, 0.1% ormore, 0.5% or more, 30% or less, 10% or less, 5% or less, 3% or less, or1% or less. The percent of cross-linking is preferably 0.01 to 30%, morepreferably 0.05 to 10%, still more preferably 0.1 to 5%, yet morepreferably 0.25 to 4%, particularly preferably 0.5 to 3%.

<Determination of Percent of Cross-Linking>

The percent of cross-linking of the CS derivative having a cross-linkedstructure through a group in a diamine can be determined by, forexample, the method described below. Specifically, dilute sulfuric acidis added to a test substance followed by being heated at 60° C. for sixhours, and the resultant solution is then basified. Propylene oxide isadded to the basified solution and heated at 60° C. overnight. Thestrongly acidified solution is heated at 110° C. overnight, and theresultant solution is basified. The amino group of the cross-linker islabeled with phenyl isothiocyanate, and the labeled cross-linker isquantified by means of LC/MS. The ratio of the amount by mole of thecross-linker to that of the disaccharide unit of CS is calculated inpercentage.

<Pharmaceutically Acceptable Carrier>

Example of the “pharmaceutically acceptable carrier” as used hereininclude saline, phosphate buffered saline, purified water, and water forinjection. The pharmaceutically acceptable carrier may contain a commonadditive, such as a pH adjuster, a buffer, an isotonizing agent, astabilizer, or a preservative. Examples of the additive include sodiumchloride, potassium chloride, sodium dihydrogen phosphate, disodiumhydrogen phosphate, monopotassium dihydrogen phosphate, sodium edetate,and benzalkonium chloride.

<Composition Containing Cross-Linked CS>

No particular limitation is imposed on the method for preparing thecomposition containing the cross-linked CS of the present invention(hereinafter may be referred to as “the composition of the presentinvention”). For example, the composition of the present invention canbe prepared by mixing the cross-linked CS of the present invention witha pharmaceutically acceptable carrier, and shaking the resultant mixtureby means of a shaking apparatus for 4 to 24 hours or longer.

The composition containing the cross-linked CS of the present inventionis preferably in the form of a liquid, more preferably in the form of anaqueous solution in view of sufficiently homogeneous dispersion of themicroparticulate solute in a solvent. As used herein, the term “aqueoussolution” refers to a composition containing water as a solvent andbeing in a clear form (hereinafter may be referred to as “solutionform”). The solution form can be determined by the “test for determiningsolution form” described below. In the case where the composition of thepresent invention is used as a pharmaceutical preparation (e.g., an eyedrop), the presence of insoluble matter in the composition may cause aforeign body. Thus, the composition of the present invention ispreferably in the form of an aqueous solution. The compositioncontaining water as a solvent is, for example, a composition prepared bymixing the cross-linked CS of the present invention with saline,phosphate buffered saline, purified water, or water for injection.

The cross-linked CS of the present invention is preferablywater-soluble. The “test for determining water solubility” describedbelow can determine whether or not the cross-linked CS is water-soluble.

In the composition containing the cross-linked CS of the presentinvention, the cross-linked CS exhibits a filter-passing rate ofpreferably 50% or more, more preferably 60% or more, still morepreferably 70% or more, yet more preferably 80% or more, particularlypreferably 85% or more.

The composition containing the cross-linked CS of the present inventionmay have a viscosity of 5 to 11,000, 10 to 5,000, 20 to 1,000, 30 to300, or 30 to 250 mPa·s. The concentration of the cross-linked CS in thecomposition containing the cross-linked CS of the present invention maybe 0.1 to 15 wt. %, 0.3 to 13 wt. %, or 1 to 10 wt. % relative to theentire amount of the composition.

<Test for Determining Solution Form>

The absorbance of a prepared composition at 600 nm is measured by meansof an ultraviolet visible spectrophotometer (UV-1800, manufactured byShimadzu Corporation). A composition exhibiting an absorbance of 0.1 orless is determined as “clear,” and a composition exhibiting anabsorbance of more than 0.1 is determined as “turbid.”

<Test for Determining Water Solubility>

A cross-linked CS is dissolved in water or PBS so as to achieve aconcentration of 2.0% (w/w), and the absorbance of the resultantcomposition at 600 nm is measured by means of an ultraviolet visiblespectrophotometer (UV-1800, manufactured by Shimadzu Corporation). Across-linked CS exhibiting an absorbance of 0.1 or less is determined as“water-soluble.”

<Measurement of Viscosity>

The viscosity of the composition is measured at 25° C. and 5 rpm bymeans of an E-type rotary viscometer (TV-L/H, Toki Sangyo Co., Ltd.)with a standard cone (CORD-1, 1°34′×R24). The measured viscosity isdefined as “viscosity” (mPa·s) as used herein. If the viscosity at 5 rpmfalls outside the range of detection, an extrapolated value isdetermined at another rotation speed, and the value is defined as“viscosity” (mPa·s) as used herein.

<Test for Filter-Passing Rate>

A sample is passed through a porous filter (pore size: 5.0 μm, diameter:25 mm) at 25° C., and the CS concentration (in terms of disaccharideunit) of the sample (hereinafter may be referred to as “CSconcentration”) is measured before and after the passage of the samplethrough the filter by the carbazole-sulfuric acid method describedbelow. The “filter-passing rate” as used herein is calculated by thefollowing formula (B). The pressure during passage of the sample throughthe filter is adjusted to 5.3 kgf/cm² or less.

[Formula 2](the CS concentration (wt. %) of a sample after passage through afilter)/(the CS concentration (wt. %) of the sample before passagethrough the filter)×100(%)  (B)

For example, when the CS concentration of a sample before passagethrough a filter is 1.00% and the CS concentration of the sample afterpassage through the filter is 0.90%, the filter-passing rate is 90%.

<Calculation of CS Concentration by the Carbazole-Sulfuric Acid Method>

The CS concentration (mol/L) is calculated by the following formula (C)in accordance with the carbazole-sulfuric acid method by use of 20.0μg/mL aqueous D-glucuronolactone (molecular weight: 176.12) solution asa standard.

[Formula 3]OD530 [sample]÷OD530 [standard]×D-glucuronolactone concentration(mol/L)  (C)(where OD530 corresponds to optical density at a wavelength of 530 nm).

The cross-linked CS of the present invention and the composition of thepresent invention can be used, for example, as a pharmaceuticalpreparation or an agent for the treatment of an eye disease. Thus, thecross-linked CS of the present invention can be used as an activepharmaceutical ingredient of a pharmaceutical preparation or an agentfor the treatment of an eye disease.

<Agent for the Treatment of Eye Disease>

The agent for the treatment of an eye disease of the present inventioncontains the cross-linked CS of the present invention as an activepharmaceutical ingredient. The cross-linked CS is any of those describedhereinabove; in particular, any of the aforementioned CS derivativesaccording to embodiments [A1] to [A22]. No particular limitation isimposed on the “eye disease” as used herein, so long as it causes anyabnormality in the anterior segment of the eye. The anterior segment ofthe eye is preferably the ocular surface, more preferably the cornea,and particularly preferably the superficial cornea. The “anteriorsegment of the eye” as used herein includes tear. The “eye disease” asused herein is preferably corneal epithelial disorder, abnormal tearfilm, or dry eye, and particularly preferably corneal epithelialdisorder or dry eye. The “dry eye” as used herein may be either or bothof dry eye disease and dry eye syndrome. The term “abnormal tear film”corresponds to the state where the tear film is broken or the tear filmis likely to be broken, and includes the state of “tear filminstability” according to the definition of dry eye by DEWS (The OcularSurface Vol. 5, No. 2: 75-92, 2007). Abnormal tear film is caused by,for example, increased tear evaporation or reduced tear volume. Abnormaltear film can be determined by, for example, the Schirmer test, breakuptime (BUT), or surface regularity index (SRI).

No particular limitation is imposed on the “corneal epithelialdisorder,” so long as it is the disorder in superficial cornea. Thecorneal epithelial disorder is, for example, corneal epithelial defect,corneal erosion, corneal ulcer, corneal perforation. Examples of thecorneal epithelial disorder include corneal epithelial disorders causedby superficial punctate keratopathy, keratitis, or the like. Otherexamples of the corneal epithelial disorder include corneal epithelialdisorders caused by endogenous diseases, such as dry eye, Sjogren'ssyndrome, and Stevens-Johnson syndrome; and corneal epithelial disorderscaused by exogenous factors, such as contact lens wear, trauma, surgery,infection, or pharmaceutical preparation. Particularly preferred is acorneal epithelial disorder caused by dry eye.

The term “treatment” as used herein includes therapy. The term “therapy”includes amelioration, healing, and healing acceleration. Thus, the term“agent for the treatment” includes a therapeutic agent, and the term“therapeutic agent” includes an ameliorating agent and a healingaccelerator. As used herein, the therapy for abnormal tear film may bereferred to as “tear film stabilization.”

The “agent for the treatment of eye disease” as used herein ispreferably an agent for the treatment of abnormality in the anteriorsegment of the eye, in particular, abnormality in the ocular surface ortear. Specific examples of the agent include an agent for the treatmentof a corneal epithelial disorder, a tear film stabilizer, and an agentfor the treatment of dry eye.

<Dosage Form and Preparation>

No particular limitation is imposed on the dosage form of the agent forthe treatment of an eye disease containing the cross-linked CS of thepresent invention as an active pharmaceutical ingredient (hereinafterthe agent may be referred to as “the agent of the present invention”).Examples of the dosage form include an eye drop, an eye ointment, acream, and a lotion. Of these, an eye drop is preferred, and an aqueouseye drop is particularly preferred. The term “aqueous eye drop” refersto an eye drop containing water in an amount of 50 wt. % or more. Theaqueous eye drop of the present invention contains water in an amount ofpreferably 80 wt. % or more, more preferably 90 wt. % or more. Theaqueous eye drop of the present invention may contain anypharmaceutically or physiologically acceptable water. Examples of thewater include distilled water, purified water, sterilized purifiedwater, water for injection, and distilled water for injection. Thesedefinitions are based on The Japanese Pharmacopoeia, Sixteenth Edition.The eye drop may contain, for example, saline or phosphate bufferedsaline. The eye drop may appropriately contain a common additive, suchas a pH adjuster, a buffer, an isotonizing agent, a stabilizer, or apreservative. Examples of the additive include sodium chloride,potassium chloride, sodium dihydrogen phosphate, disodium hydrogenphosphate, monopotassium dihydrogen phosphate, sodium edetate, andbenzalkonium chloride. No particular limitation is imposed on the pH andosmotic pressure ratio of the eye drop, so long as they fall withinranges acceptable for ophthalmic preparations. The agent of the presentinvention does not necessarily contain hyaluronic acid.

The agent of the present invention is preferably in the form of aliquid, more preferably in the form of an aqueous solution in view ofsufficiently homogeneous dispersion of the microparticulate solute in asolvent. In the case where the agent of the present invention is used asan eye drop, the presence of insoluble matter in the agent may cause aforeign body sensation. Thus, the agent of the present invention ispreferably in the form of an aqueous solution. In the agent of thepresent invention, the cross-linked CS exhibits a filter-passing rate ofpreferably 60% or more, more preferably 70% or more, still morepreferably 80% or more, particularly preferably 85% or more.

No particular limitation is imposed on the concentration of thecross-linked CS in the agent of the present invention, so long as it isan effective concentration exhibiting a desired medical efficacy. Theconcentration of the cross-linked CS is, for example, 0.01 to 20 wt. %,0.1 to 15 wt. %, or 0.3 to 10 wt. %.

<Application Target>

The application target of the agent of the present invention ispreferably a mammal. Examples of the mammal include, but are notparticularly limited to, human, equine, bovine, canine, feline, rabbit,hamster, guinea pig, and mouse. The agent of the present invention canserve as a pharmaceutical preparation for a human or an animal,preferably a pharmaceutical preparation for a human

<Usage and Dosage>

The dosage of the agent of the present invention can be appropriatelyvaried depending on, for example, the degree of patient's symptom, theage or body weight of a patient, or the diagnosis by a physician.

No particular limitation is imposed on the number of instillation timesof the agent of the present invention per day or the instillationperiod. The number of instillation times of the agent per day is, forexample, one to eight, one to six, one to four, one to three, one, ortwo, and the number may be “appropriately increased or decreased. Noparticular limitation is imposed on the instillation period, and theperiod is, for example, one week to several months. The agent ispreferably instilled every day. For the case of an eye drop, forexample, one or two drops or one to three drops are instilled withsingle application; specifically, one to eight ocular instillations perday (one to three drops per instillation), one to six ocularinstillations per day (one to three drops per instillation), one to fourocular instillations per day (one to three drops per instillation), oneto three ocular instillations per day (one to three drops perinstillation), or one or two ocular instillations per day (one to threedrops per instillation). For example, the eye drop is instilled everyday.

<Method of Using the Agent of the Present Invention>

The agent of the present invention can be instilled into the eyes of ahuman or an animal. No particular limitation is imposed on theinstillation of the agent of the present invention into the eye(s) of ahuman or an animal, so long as the agent is instilled in a medicallyacceptable manner so that the advantageous effects of the presentinvention are achieved.

No particular limitation is imposed on the specific manner ofinstillation of the agent. The instillation manner may be appropriatelydetermined depending on the form of dosage or preparation, and ispreferably, for example, ocular instillation.

<Method for Treatment of Eye Disease>

The treatment method of the present invention is a method for thetreatment of an eye disease, the method involving instillation of theagent of the present invention or the composition containing thecross-linked CS of the present invention into the eyes of a human or ananimal. The treatment method of the present invention can be carried outin the same manner as described above, for example, in the sections<application target>, <usage and dosage>, and <method of using the agentof the present invention>.

EXAMPLES

The present invention will next be described in more detail by way ofexamples and test examples, which should not be construed as limitingthe technical scope of the invention thereto. Unless otherwisespecified, the “%” is on a mass basis. Unless otherwise specified, themole equivalent (represented by “eq”) of a polyvalent amine as across-linking agent and a condensing agent is relative to thedisaccharide unit of CS.

Example 1

Production of Cross-Linked CS—NC2N

(A) Typical Example

CS (CSC, sodium chondroitin sulfate (Japanese Pharmaceutical Codex),weight average molecular weight: 40,000, Seikagaku Corporation) (2.00 g)(disaccharide unit: 3.91 mmol [calculated from the average molecularweight of the disaccharide unit (=511)], 1.00 eq) was dissolved in waterfor injection (WFI) so as to achieve a concentration of 10%. Theresultant solution was mixed with ethanol (EtOH) (20.0 mL). A solution(4.00 mL) of ethane-1,2-diamine dihydrochloride (NC2N.2HCl, Wako PureChemical Industries, Ltd.) (i.e., a cross-linking agent) (10.4 mg,0.0783 mmol, 0.0200 eq) in 50% ethanol and a solution (4.00 mL) ofDMT-MM (TRIAZIMOCH, Tokuyama Corporation) (i.e., a condensing agent)(86.6 mg, 0.313 mmol, 0.0800 eq) in 50% ethanol were sequentially addeddropwise to the mixture, and the resultant mixture was agitated at roomtemperature overnight (cross-linking reaction). 10% Aqueous sodiumcarbonate solution (Na₂CO₃/WFI) (20 mL) was added to the mixture, andthe mixture was vigorously agitated for two to four hours. The resultantmixture was neutralized with 50 wt. % aqueous acetic acid solution(AcOH/WFI) (4.0 mL), and then sodium chloride (NaCl) (2.0 g) was addedto the mixture, followed by precipitation with EtOH. The resultantprecipitate was collected through filtration and washed with 90% EtOH(200 mL) thrice and then with EtOH (200 mL) twice. The resultantprecipitate was dried under reduced pressure at 42° C. overnight, tothereby produce a cross-linked CS product (cross-linked CS—NC2N,compound 1) as a white powder (1.81 g).

(B) Production Example Different from Typical Example in Terms ofEquivalent of Cross-Linking Agent

The procedure of Example 1(A) was repeated, except that the moleequivalent of NC2N.2HCl was changed to 0.0050, 0.0100, 0.0150, 0.0175,0.0225, 0.0250, and 0.0275 eq, and the mole equivalent of DMT-MM waschanged to 0.0200, 0.0400, 0.0600, 0.0700, 0.0900, 0.100, and 0.110 eq,to thereby produce cross-linked CS products (cross-linked CS—NC2N,compounds 2 to 8) as white powders (about 1.75 g each).

Example 2

Production of Cross-Linked CS—NC2N

(A) 10 g Scale

The procedure of Example 1(A) was repeated, except that the amount of CSwas changed to 10 g, the mole equivalent of NC2N.2HCl was changed to0.020 eq, and the mole equivalent of DMT-MM was changed to 0.080 eq, tothereby produce a cross-linked CS product (cross-linked CS—NC2N,compound 9) as a white powder (8.94 g).

(B) 20 g Scale

The procedure of Example 1(A) was repeated, except that the amount of CSwas changed to 20 g, the mole equivalent of NC2N.2HCl was changed to0.02375 eq, the mole equivalent of DMT-MM was changed to 0.0950 eq, andthe reaction time of cross-linking was changed to 3 hours and 46minutes, to thereby produce a cross-linked CS product (cross-linkedCS—NC2N, compound 10) as a white powder (19.1 g).

Example 3

Production of Cross-Linked CS—NC3N

The procedure of Example 1(A) was repeated, except that the amount of CSwas changed to 1 g, 1,3-diaminopropane dihydrochloride (NC3N.2HCl, TokyoChemical Industry Co., Ltd.) was used as a cross-linking agent (moleequivalent: 0.0150, 0.0200, 0.0300, and 0.0400 eq), and the moleequivalent of DMT-MM was changed to 0.0600, 0.0800, 0.120, and 0.160 eq,to thereby produce cross-linked CS products (cross-linked CS—NC3N,compounds 11 to 14) as white powders (about 0.84 g each).

Example 4

Production of Cross-Linked CS—NC4N

The procedure of Example 1(A) was repeated, except that the amount of CSwas changed to 4 g, 1,4-diaminobutane dihydrochloride (NC4N.2HCl, WakoPure Chemical Industries, Ltd.) was used as a cross-linking agent (moleequivalent: 0.0300, 0.0425, 0.0450, and 0.0475 eq), and the moleequivalent of DMT-MM was changed to 0.120, 0.170, 0.180, and 0.190 eq,to thereby produce cross-linked CS products (cross-linked CS—NC4N,compounds 15 to 18) as white powders (about 3.85 g each).

Example 5

Production of Cross-Linked CS—NC5N

The procedure of Example 1(A) was repeated, except that the amount of CSwas changed to 1 g, 1,5-diaminopentane dihydrochloride (NC5N.2HCl, TokyoChemical Industry Co., Ltd.) was used as a cross-linking agent (moleequivalent: 0.0250, 0.0350, 0.0450, and 0.0550 eq), and the moleequivalent of DMT-MM was changed to 0.100, 0.140, 0.180, and 0.220 eq,to thereby produce cross-linked CS products (cross-linked CS—NC5N,compounds 19 to 22) as white powders (about 0.85 g each).

Example 6

Production of Cross-Linked CS—NC6N

The procedure of Example 1(A) was repeated, except that the amount of CSwas changed to 4 g, 1,6-diaminohexane dihydrochloride (NC6N.2HCl, WakoPure Chemical Industries, Ltd.) was used as a cross-linking agent (moleequivalent: 0.0400, 0.0525, 0.0550, and 0.0575 eq), and the moleequivalent of DMT-MM was changed to 0.160, 0.210, 0.220, and 0.230 eq,to thereby produce cross-linked CS products (cross-linked CS—NC6N,compounds 23 to 26) as white powders (about 3.83 g each).

Example 7

Production of Cross-Linked CS—NC8N

The procedure of Example 1(A) was repeated, except that the amount of CSwas changed to 4 g, 1,8-diaminooctane dihydrochloride (NC8N.2HCl,prepared by addition of 1N hydrochloric acid (2.00 eq) to1,8-diaminooctane [Wako Pure Chemical Industries, Ltd.]) was used as across-linking agent (mole equivalent: 0.0500, 0.0650, 0.0700, and 0.0725eq), and the mole equivalent of DMT-MM was changed to 0.200, 0.260,0.280, and 0.290 eq, to thereby produce cross-linked CS products(cross-linked CS—NC8N, compounds 27 to 30) as white powders (about 3.88g each).

Example 8

Production of Cross-Linked CS—NC12N

The procedure of Example 1(A) was repeated, except that the amount of CSwas changed to 1 g, 1,12-diaminododecane dihydrochloride (NC12N.2HCl,prepared by addition of 1N hydrochloric acid (2.00 eq) to1,12-diaminododecane [Sigma-Aldrich]) was used as a cross-linking agent(mole equivalent: 0.0550 and 0.0700 eq), and the mole equivalent ofDMT-MM was changed to 0.220 and 0.280 eq, to thereby producecross-linked CS products (cross-linked CS—NC12N, compounds 31 and 32) aswhite powders (about 0.82 g each).

Example 9

Production of Cross-Linked CS-LysEt

The procedure of Example 1(A) was repeated, except that the amount of CSwas changed to 1 g, L-lysine ethyl ester dihydrochloride (LysEt.2HCl,Sigma-Aldrich) was used as a cross-linking agent (mole equivalent:0.0450 eq), the mole equivalent of DMT-MM was fixed to 0.180 eq, and thereaction time of cross-linking was changed to 2 hours, 3 hours, 3 hoursand 40 minutes, 4 hours and 20 minutes, 5 hours, and 5 hours and 50minutes, to thereby produce cross-linked CS products (cross-linkedCS-LysEt, compounds 33 to 38) as white powders (about 1.04 g each).

Example 10

Production of Cross-Linked CS-OrnEt

The procedure of Example 1(A) was repeated, except that the amount of CSwas changed to 2 g, ornithine ethyl ester dihydrochloride (OrnEt.2HCl,Chem-Impex International) was used as a cross-linking agent (moleequivalent: 0.0200, 0.0300, 0.0325, and 0.0350 eq), and the moleequivalent of DMT-MM was changed to 0.0800, 0.120, 0.130, and 0.140 eq,to thereby produce cross-linked CS products (cross-linked CS-OrnEt,compounds 39 to 42) as white powders (about 1.99 g each).

Example 11

Production of Cross-Linked CS-Spermidine

The procedure of Example 1(A) was repeated, except that the amount of CSwas changed to 1 or 2 g, spermidine trihydrochloride (Spermidine.3HCl,Sigma-Aldrich) was used as a cross-linking agent (mole equivalent:0.0100, 0.0200, and 0.0220 eq), and the mole equivalent of DMT-MM waschanged to 0.0400, 0.0800, and 0.0880 eq, to thereby producecross-linked CS products (cross-linked CS-Spermidine, compounds 43, 44,and 67) as white powders (about 0.77 g, about 0.77 g, and about 1.75 g,respectively).

Example A1

Production of Cross-Linked CS-triAmine

The procedure of Example 1(A) was repeated, except that the amount of CSwas changed to 2 g, 2-(aminomethyl)-2-methylpropane-1,3-diaminetrihydrochloride (triAmine.3HCl, Aldrich) was used as a cross-linkingagent (mole equivalent: 0.0091, 0.0092, and 0.0097 eq), and the moleequivalent of DMT-MM was changed to 0.0364, 0.0368, and 0.0388 eq, tothereby produce cross-linked CS products (cross-linked CS-triAmine,compounds 48 to 50) as white powders (about 1.74 g each).

Example A2

Production of Cross-Linked CS—NC3(OH)N

The procedure of Example 1(A) was repeated, except that the amount of CSwas changed to 2 g, 1,3-diamino-2-propanol dihydrochloride(NC3(OH)N.2HCl, prepared by addition of 1N hydrochloric acid (2.00 eq)to 1,3-diamino-2-propanol [Aldrich]) was used as a cross-linking agent(mole equivalent: 0.0220, 0.0240, and 0.0260 eq), and the moleequivalent of DMT-MM was changed to 0.0880, 0.0960, and 0.1040 eq, tothereby produce cross-linked CS products (cross-linked CS—NC3(OH)N,compounds 51 to 53) as white powders (about 1.75 g each).

Example A3

Production of Cross-Linked CS—NC4(═)N

The procedure of Example 1(A) was repeated, except that the amount of CSwas changed to 2 g, (E)-2-butene-1,4-diamine dihydrochloride(NC4(═)N.2HCl, Small Molecules, Inc.) was used as a cross-linking agent(mole equivalent: 0.0270 and 0.0275 eq), and the mole equivalent ofDMT-MM was changed to 0.1080 and 0.1100 eq, to thereby producecross-linked CS products (cross-linked CS—NC4(═)N, compounds 54 and 55)as white powders (about 1.80 g each).

Example A4

Production of Cross-Linked CS-Xylylene

The procedure of Example 1(A) was repeated, except that the amount of CSwas changed to 2 g, 1,4-bis(aminomethyl)benzene dihydrochloride(Xylylene.2HCl, prepared by addition of 1N hydrochloric acid (2.00 eq)to 1,4-bis(aminomethyl)benzene [Aldrich]) was used as a cross-linkingagent (mole equivalent: 0.0135, 0.0145, and 0.0150 eq), and the moleequivalent of DMT-MM was changed to 0.0540, 0.0580, and 0.0600 eq, tothereby produce cross-linked CS products (cross-linked CS-Xylylene,compounds 56 to 58) as white powders (about 1.77 g each).

Example A5

Production of Cross-Linked CS-Cyclohex

The procedure of Example 1(A) was repeated, except that the amount of CSwas changed to 2 g, 1,4-bis(aminomethyl)cyclohexane dihydrochloride(Cyclohex.2HCl, prepared by addition of 1N hydrochloric acid (2.00 eq)to 1,4-bis(aminomethyl)cyclohexane [Kanto Chemical Co., Inc.]) was usedas a cross-linking agent (mole equivalent: 0.0410 and 0.0425 eq), andthe mole equivalent of DMT-MM was changed to 0.1640 and 0.1700 eq, tothereby produce cross-linked CS products (cross-linked CS-Cyclohex,compounds 59 and 60) as white powders (about 1.80 g each).

Example A6

Production of Cross-Linked CS—NC5(S)N

The procedure of Example 1(A) was repeated, except that the amount of CSwas changed to 2 g, 2,2′-thiodiethanamine dihydrochloride (NC5(S)N.2HCl,prepared by addition of 1N hydrochloric acid (2.00 eq) to2,2′-thiodiethanamine [Tokyo Chemical Industry Co., Ltd.]) was used as across-linking agent (mole equivalent: 0.0170 and 0.0180 eq), and themole equivalent of DMT-MM was changed to 0.0680 and 0.0720 eq, tothereby produce cross-linked CS products (cross-linked CS—NC5(S)N,compounds 61 and 62) as white powders (about 1.77 g each).

Example A7

Production of Cross-Linked CS-Glycol(C5)

The procedure of Example 1(A) was repeated, except that the amount of CSwas changed to 2 g, 2,2′-oxydiethanamine dihydrochloride(Glycol(C5).2HCl, Wako Pure Chemical Industries, Ltd.) was used as across-linking agent (mole equivalent: 0.0290 and 0.0310 eq), and themole equivalent of DMT-MM was changed to 0.1160 and 0.1240 eq, tothereby produce cross-linked CS products (cross-linked CS-Glycol(C5),compounds 63 and 64) as white powders (about 1.76 g each).

Example A8

Production of Cross-Linked CS-Glycol(C11)

The procedure of Example 1(A) was repeated, except that the amount of CSwas changed to 2 g, 1,11-diamino-3,6,9-trioxaundecane dihydrochloride(Glycol(C11).2HCl, prepared by addition of 1N hydrochloric acid (2.00eq) to 1,11-diamino-3,6,9-trioxaundecane [Tokyo Chemical Industry Co.,Ltd.]) was used as a cross-linking agent (mole equivalent: 0.0380 and0.0410 eq), and the mole equivalent of DMT-MM was changed to 0.1520 and0.1640 eq, to thereby produce cross-linked CS products (cross-linkedCS-Glycol(C11), compounds 65 and 66) as white powders (about 1.87 geach).

Comparative Example 1

Production of CS—NC12N Derivative (Comparative Example)

A powder of a CS derivative modified with 1,12-diaminododecane wasproduced through the method described below. According to the literaturefrom which the production method is cited, the modification is carriedout for reducing the hydrophilicity of CS.

Through the method described in Example 5 “A. Modification ofchondroitin” of WO 91/16881 and Biomaterials, 16: 473-478, 1995, CSderivatives (“CS—NC12N derivatives (Comparative Examples),” compounds 45to 47) were produced as white powders (about 0.46 g each) by use ofchondroitin sulfate A (CSA, Sigma-Aldrich) (1.00 g) (disaccharide unit:2.18 mmol [calculated from the average molecular weight of thedisaccharide unit (=458)], 1.00 eq), 1,12-diaminododecanedihydrochloride (Sigma-Aldrich) (mole equivalent: 0.30, 0.60, and 0.90eq), and dicyclohexylcarbodiimide (DCC) serving as a condensing agent(mole equivalent: 0.66, 1.32, and 1.98 eq).

Tables 1 to 4 illustrate the compounds produced in the aforementionedExamples and Comparative Examples.

TABLE 1 Equivalent of cross-linking Equivalent agent of DMT-MM CompoundCross-linked product Cross-linking agent (eq) (eq) Compound 1Cross-linked CS-NC2N NC2N•2HCl 0.0200 0.0800 Compound 2 (Example 1, 2)0.0050 0.0200 Compound 3 0.0100 0.0400 Compound 4 0.0150 0.0600 Compound5 0.0175 0.0700 Compound 6 0.0225 0.0900 Compound 7 0.0250 0.100Compound 8 0.0275 0.110 Compound 9 0.020 0.080 Compound 10 0.023750.0950 Compound 11 Cross-linked CS-NC3N NC3N•2HCl 0.0150 0.0600 Compound12 (Example 3) 0.0200 0.0800 Compound 13 0.0300 0.120 Compound 14 0.04000.160 Compound 15 Cross-linked CS-NC4N NC4N•2HCl 0.0300 0.120 Compound16 (Example 4) 0.0425 0.170 Compound 17 0.0450 0.180 Compound 18 0.04750.190 Compound 19 Cross-linked CS-NC5N NC5N•2HCl 0.0250 0.100 Compound20 (Example 5) 0.0350 0.140 Compound 21 0.0450 0.180 Compound 22 0.05500.220 Compound 23 Cross-linked CS-NC6N NC6N•2HCl 0.0400 0.160 Compound24 (Example 6) 0.0525 0.210 Compound 25 0.0550 0.220 Compound 26 0.05750.230 Compound 27 Cross-linked CS-NC8N NC8N•2HCl 0.0500 0.200 Compound28 (Example 7) 0.0650 0.260 Compound 29 0.0700 0.280 Compound 30 0.07250.290 Compound 31 Cross-linked CS-NC12N NC12N•2HCl 0.0550 0.220 Compound32 (Example 8) 0.0700 0.280

TABLE 2 Equivalent of cross-linking Equivalent agent of DMT-MM CompoundCross-linked product Cross-linking agent (eq) (eq) Compound 33¹⁾Cross-linked CS-LysEt LysEt•2HCl 0.0450 0.180 Compound 34¹⁾ (Example 9)Compound 35¹⁾ Compound 36¹⁾ Compound 37¹⁾ Compound 38¹⁾ Compound 39Cross-linked CS-OrnEt OrnEt•2HCl 0.0200 0.0800 Compound 40 (Example 10)0.0300 0.120 Compound 41 0.0325 0.130 Compound 42 0.0350 0.140 Compound43 Cross-linked CS- Spermidine•3HCl 0.0100 0.0400 Compound 44 Spermidine0.0200 0.0800 (Example 11)1) Compounds 33 to 38 were produced under the following conditions: theequivalent of the cross-linking agent (fixed); the equivalent of DMT-MM(fixed); and reaction time of cross-linking: 2 hours (compound 33), 3hours (compound 34), 3 hours and 40 minutes (compound 35), 4 hours and20 minutes (compound 36), 5 hours (compound 37), and 5 hours and 50minutes (compound 38).

TABLE 3 Equivalent of cross-linking Equivalent agent of DMT-MM CompoundCross-linked product Cross-linking agent (eq) (eq) Compound 48Cross-linked CS- triAmine•3HCl 0.0091 0.0364 Compound 49 triAmine 0.00920.0368 Compound 50 (Example A1) 0.0097 0.0388 Compound 51 Cross-linkedCS- NC3(OH)N•2HCl 0.0220 0.0880 Compound 52 NC3(OH)N 0.0240 0.0960Compound 53 (Example A2) 0.0260 0.1040 Compound 54 Cross-linked CS-NC4(═)N•2HCl 0.0270 0.1080 Compound 55 NC4(═)N 0.0275 0.1100 (ExampleA3) Compound 56 Cross-linked CS- Xylylene•2HCl 0.0135 0.0540 Compound 57Xylylene 0.0145 0.0580 (Example A4) Compound 58 0.0150 0.0600 Compound59 Cross-linked CS- Cyclohex•2HCl 0.0410 0.1640 Compound 60 Cyclohex0.0425 0.1700 (Example A5) Compound 61 Cross-linked CS- NC5(S)N•2HCl0.0170 0.0680 Compound 62 NC5(S)N 0.0180 0.0720 (Example A6) Compound 63Cross-linked CS- Glycol(C5)•HCl 0.0290 0.1160 Compound 64 Glycol(C5)0.0310 0.1240 (Example A7) Compound 65 Cross-linked CS- Glycol(C11)•2HCl0.0380 0.1520 Compound 66 Glycol(C11) 0.0410 0.1640 (Example A8)Compound 67 Cross-linked CS- Spermidine•3HCl 0.0220 0.0880 Spermidine(Example 11)

TABLE 4 Equivalent of cross-linking Equivalent Cross-linking agent ofDCC Compound Derivative agent (eq) (eq) Compound 45 CS-NC12N DerivativeNC12N•2HCl 0.30 0.66 Compound 46 (Comparative Example) 0.60 1.32Compound 47 (Comparative Example 1) 0.90 1.98

Example 12A

Analysis (Determination of Cross-Linked Structure)

The CS—NC12N derivative produced through the method described in WO91/16881 was analyzed by various analytic methods. However, thecross-linkage of CS was not determined (C. Bourie, et al., J. Biomater.Appl., 12, (1998), 201-221). For determination of the cross-linkage ofthe cross-linked CS of the present invention, the cross-linked CS wasdigested with an enzyme, and the digested product was analyzed by meansof liquid chromatography (HPLC)/multi-stage mass spectrometry.

<Test Substance>

CS (CSC, sodium chondroitin sulfate (Japanese Pharmaceutical Codex),weight average molecular weight: 40,000, Seikagaku Corporation) and thecross-linked CS—NC2N (compound 9) were used.

<Method>

(1) Enzymatic Digestion of CS and Cross-Linked CS—NC2N

Chondroitinase ABC (C-ABC) (Seikagaku Corporation) was diluted with 50mM tris-HCl buffer (pH 7.5)-0.1% BSA solution (50 U/mL). The dilutedC-ABC (10 μl) was added to 1% w/v aqueous CS solution (20 μL) or 1% w/vaqueous cross-linked CS—NC2N solution (20 μl). The resultant mixture washeated at 37° C. for three hours and then boiled for 30 seconds, tothereby stop the reaction.

(2) HPLC Analysis of Enzyme-Digested Product

The C-ABC-digested liquid sample was diluted to prepare a solution (200μL in total), the liquid comprising water and acetonitrile (=1:1 (v/v)).The resultant solution was analyzed by means of an LC-MS apparatus ofHPLC (Prominence, manufactured by Shimadzu Corporation) connected toESI-MS (LCMS-IT-TOF, manufactured by Shimadzu Corporation). TSKgelAmide-80 HR (4.6 mm I.D.×250 mm) was used as a column. 20 mM aqueousammonium formate solution (Solvent A) and acetonitrile (Solvent B) wereused as eluents for analysis of the C-ABC-digested liquid sample(detection: 232 nm and MS). ESI-MS was operated under the followingconditions: interface voltage: −3.5 kV, CDL temperature: 200° C., heatblock temperature: 200° C., scanning molecular weight range: m/z=300 to1,500 for MS, m/z=50 to 2,000 for MS^(n).

(3) Multi-Stage Mass Spectrometry of Newly Observed Peak

Peaks observed only in the chart of the cross-linked CS—NC2N throughLC-MS analysis were subjected to mass spectrometry under the conditionsdescribed below. LCMS-IT-TOF (manufactured by Shimadzu Corporation)connected to a nanoESI ion source (NES-100, manufactured by NewObjective) was used for ESI-MS. A solution containing the cross-linkedCS—NC2N was fractionated under the aforementioned HPLC conditions, andthe solvent was removed from the solution under reduced pressure.Thereafter, the residue was dissolved in a 0.5% NH₃-containing 50% MeOHsolution. The resultant solution was directly injected by means of asyringe pump. ESI-MS was operated under the following conditions: flowrate: 10 μL/h, interface voltage: −1.5 kV, CDL temperature: 200° C.,heat block temperature: 200° C., scanning molecular weight range:m/z=1,300 to 1,500 for MS, m/z=50 to 2,000 for MS^(n).

<Test Results>

The HPLC charts of the digested liquids illustrate that CS and thecross-linked CS—NC2N were similarly digested with C-ABC (FIG. 1). PeaksA and B in FIG. 1 are typical peaks that are not observed in the HPLCchart of non-cross-linked CS. The LC-MS analysis suggested that thesepeaks belong to a structure having a group in a diamine. Table 5summarizes structures that are presumed to have a group in a diamine onthe basis of mass-to-charge ratio.

Peak B was subjected to multi-stage mass spectrometry for determining astructure cross-linked with a group in a diamine. FIGS. 2 and 3illustrate some of the results. As illustrated in FIGS. 2 and 3, peakssuggesting the covalent bonding between ethane-1,2-diamine and twoglucuronic acid molecules of CS were detected.

TABLE 5 Composition m/z Valence M GalNAc GlcA Sulfate group CrosslinkerNa Theoretical value Error (ppm) 710.60 2 1423.22 3 3 3 1 1 1423.23−9.84 524.75 3 1577.27 3 4 3 1 0 1577.28 −3.80 532.08 3 1599.26 3 4 3 11 1599.26 1.25 592.44 3 1780.34 4 4 3 1 0 1780.36 −8.43 464.07 4 1860.314 4 4 1 0 1860.32 −1.61 660.40 3 1984.22 4 4 5 1 2 1984.24 −5.54 806.123 2421.38 5 5 6 1 1 2421.32 26.43

<Conclusion>

The liquid chromatography/multi-stage mass spectrometry of theenzyme-digested cross-linked CS—NC2N showed the covalent bonding betweenthe diamine and the carboxy groups of two molecules of glucuronic acid.The results demonstrated that the cross-linked CS of the presentinvention has a cross-linked structure.

Example 12B

Determination of Percent of Cross-Linking

A portion of the sample prepared in Example 1 or 2 was weighed (10 mg)and dissolved in 20 mM sulfuric acid (1 mL) followed by subjected todegassing. The resultant solution was heated at 60° C. for six hours andthen basified. Ethanol and propylene oxide were added to the basifiedsolution, and the solution was heated at 60° C. overnight. The solutionwas cooled and acidified followed by subjected to evaporation to drynessunder reduced pressure. Diaminobutane (internal standard) was added tothe resultant product, and 6 M hydrochloric acid was added thereto. Theresultant solution was heated at 110° C. overnight followed by subjectedto evaporation to dryness under reduced pressure. A mixture of dimethylsulfoxide and water (=3:1) was added to the resultant product, and themixture was shaken for 30 minutes. The resultant mixture was transferredto another container, and triethylamine and a mixture of phenylisothiocyanate and acetonitrile (=1:9) were added to the mixture. Themixture was vigorously agitated and then heated at 30° C. for 20minutes. The mixture was passed through a 0.22-μm filter, and thensubjected to analysis by means of LC-ESI-MS (positive mode). Gemini-NX 3μm C18 2×50 mm was used as a column. H₂O (Solvent A), MeCN (Solvent B),and 100 mM NH₄HCO₃ (pH 10.0 by NH₄OH) (Solvent C) were used as eluentsfor analysis. The results are illustrated in Table 6.

TABLE 6 Equivalent of cross- Percent of cross- Compound Cross-linkedproduct linking agent (eq) linking (%) Compound 2 Cross-linked CS-NC2N0.0050 0.064 Compound 10 0.02375 0.59 Compound 8 0.0275 0.89

Example 13

Preparation of Composition Containing Cross-Linked CS

Phosphate buffered saline (may be referred to as “PBS” herein) havingthe following composition was prepared:

0.065% (w/v) sodium dihydrogen phosphate dihydrate

0.03% (w/v) disodium hydrogen phosphate dodecahydrate

0.9% (w/v) sodium chloride.

The cross-linked CS products produced in Examples 1, 2, 4, 6 to 11, andA1 to A8 were mixed with the aforementioned PBS so as to achieveconcentrations as shown in Tables 7, 8, and 9, followed by shaking witha shaking apparatus overnight, to thereby prepare samples (samples 1 to43, 55 to 71, and 75 to 77). Thereafter, some samples were measured forabsorbance at 600 nm by means of an ultraviolet visiblespectrophotometer (UV-1800, manufactured by Shimadzu Corporation). Theresults are illustrated in Tables 7, 8, and 9.

Comparative Example 2

Preparation of Composition Containing CS

In the same manner as described in Example 13, CS (CSC [SeikagakuCorporation] and CSA [Sigma-Aldrich]) was mixed with the PBS so as toachieve concentrations as shown in Table 10, to thereby prepare samples(samples 47 to 54). Thereafter, sample 53 was measured for absorbance at600 nm by means of an ultraviolet visible spectrophotometer (UV-1800,manufactured by Shimadzu Corporation). The results are illustrated inTable 10.

Comparative Example 3

Preparation of Composition Containing CS—NC12N Derivative (ComparativeExample)

In the same manner as described in Example 13, the CS—NC12N derivative(Comparative Example) produced in Comparative Example 1 was mixed withthe PBS so as to achieve concentrations as shown in Table 8, to therebyprepare samples (samples 44 to 46 and 72 to 74). Thereafter, the sampleswere measured for absorbance at 600 nm by means of an ultravioletvisible spectrophotometer (UV-1800, manufactured by ShimadzuCorporation). The results are illustrated in Table 8.

Example 14

Measurement of Viscosities of Samples

The viscosities (mPa·s) of the samples prepared in Example 13 andComparative Example 2 were measured at 25° C. and 5 rpm by means of anE-type rotary viscometer (TV-L/H, Toki Sangyo Co., Ltd.) with a standardcone (CORD-1, 1° 34′×R24). If the viscosity at 5 rpm fell outside therange of detection, an extrapolated value was determined at anotherrotation speed, and the value was defined as viscosity (mPa·s). Theresults are illustrated in Tables 7 to 10.

TABLE 7 Concentration of cross-linked Viscosity Absorbance Solution formSample Compound CS (wt %) (mPa · s) (Abs) (clear/turbid) Sample 1Compound 2 2.00 7 — — Sample 2 3.82 18 — — Sample 3 6.00 58 — — Sample 48.00 132 — — Sample 5 10.00 274 — — Sample 6 12.00 493 — — Sample 714.00 936 0.011 clear Sample 8 Compound 3 2.00 8 — — Sample 9 4.00 29 —— Sample 10 6.00 83 — — Sample 11 8.00 199 — — Sample 12 10.00 441 — —Sample 13 12.00 890 0.007 clear Sample 14 Compound 4 2.00 12 — — Sample15 5.00 94 — — Sample 16 10.00 1245 0.014 clear Sample 17 Compound 52.00 15 — — Sample 18 5.00 161 — — Sample 19 10.03 2360 0.013 clearSample 20 Compound 1 1.97 32 — — Sample 21 5.00 367 — — Sample 22 10.003000 0.010 clear Sample 23 Compound 6 1.00 33 — — Sample 24 2.03 115 — —Sample 25 5.00 1306 — — Sample 26 7.49 4340 0.025 clear Sample 27 0.5025 — — Sample 28 Compound 7 1.00 156 — — Sample 29 2.00 673 — — Sample30 4.00 2880 — — Sample 31 6.00 8190 0.020 clear Sample 32 Compound 80.50 139 — — Sample 33 1.00 646 — — Sample 34 2.00 2800 — — Sample 354.00 10649 0.020 clear Sample 36 Compound 10 2.00 52 0.009 clear —: Nodata

TABLE 8 Concentration of cross-linked CS/derivative Viscosity AbsorbanceSolution form Sample Compound (wt %) (mPa · s) (Abs) (clear/turbid)Sample 37 Compound 18 8.00 3770 0.025 clear Sample 38 Compound 26 8.754000 0.033 clear Sample 39 Compound 30 8.00 3330 0.018 clear Sample 40Compound 32 5.00 3740 0.014 clear Sample 41 Compound 36 7.50 3600 — —Sample 42 Compound 42 8.25 3200 0.028 clear Sample 43 Compound 44 7.503230 — — Sample 44 Compound 45 2.00 — 2.923 turbid Sample 45 Compound 462.00 — 2.034 turbid Sample 46 Compound 47 2.00 — 1.457 turbid Sample 72Compound 45 0.40 — 1.352 turbid Sample 73 Compound 46 0.40 — 0.417turbid Sample 74 Compound 47 0.40 — 0.238 turbid —: No data

TABLE 9 Concentration of cross-linked Viscosity Absorbance Solution formSample Compound CS (wt %) (mPa · s) (Abs) (clear/turbid) Sample 55Compound 48 2.00 38 0.003 clear Sample 56 Compound 49 2.00 60 0.008clear Sample 57 Compound 50 2.00 122 0.001 clear Sample 58 Compound 512.00 27 0.005 clear Sample 59 Compound 52 2.00 62 0.000 clear Sample 60Compound 53 2.00 130 0.002 clear Sample 61 Compound 54 2.00 42 0.004clear Sample 62 Compound 55 2.00 98 0.003 clear Sample 63 Compound 562.00 45 0.001 clear Sample 64 Compound 57 2.00 72 0.003 clear Sample 65Compound 58 2.00 191 0.004 clear Sample 66 Compound 59 2.00 49 0.002clear Sample 67 Compound 60 2.00 88 0.001 clear Sample 68 Compound 612.00 44 0.000 clear Sample 69 Compound 62 2.00 100 0.001 clear Sample 70Compound 63 2.00 50 0.002 clear Sample 71 Compound 64 2.00 123 0.005clear Sample 75 Compound 65 2.00 51 0.002 clear Sample 76 Compound 662.00 157 0.010 clear Sample 77 Compound 67 2.00 85 0.003 clear

TABLE 10 Concentration of Viscosity Absorbance Solution form SampleCompound CS (wt %) (mPa · s) (Abs) (clear/turbid) Sample 47 CS 2.00 5 —— Sample 48 4.00 18 — — Sample 49 6.00 47 — — Sample 50 8.00 100 — —Sample 51 10.00 200 — — Sample 52 12.49 423 — — Sample 53 14.99 8220.002 clear Sample 54 CSA 2.00 — — — —: No data

<Conclusion>

Samples 7, 13, 16, 19, 22, 26, 31, 35 to 40, 42, 53, 55 to 71, and 75 to77 were found to be in a clear solution form (i.e., these samples werean aqueous solution). When a composition of higher concentration is in aclear solution form, a composition of lower concentration is alsoprobably in a clear solution form. Samples 1 to 43, 47 to 71, and 75 to77 were confirmed to be clear through visual observation (the term“clear” as used herein refers to a clear composition). In contrast,compositions containing the CS—NC12N derivative (Comparative Example)(compounds 45 to 47) were found to be in a turbid solution form (i.e.,these compositions were not an aqueous solution).

Example 15

Test for Filter-Passing Rate

Some of the samples prepared in Example 13 were subjected to the testfor filter-passing rate. Specifically, a sample was charged into a 1 mLsyringe (SS-01T, Terumo Corporation) equipped with a porous filter (poresize: 5.0 μm, diameter: 25 mm, Millex (registered trademark)-SV 5.00 μm(Millipore Ireland)). The sample was extruded with a piston at 25° C.and a pressure of 5.3 kgf/cm² or less, to thereby pass the samplethrough the filter. The CS concentration of the sample was determinedbefore passage of the sample through the filter. Also, the CSconcentration of the sample was determined after passage of the sample(in an amount of 0.5 mL or more) through the filter. The CSconcentration was determined by the carbazole-sulfuric acid method using20.0 μg/mL aqueous D-glucuronolactone solution as a standard. The ratioof the CS concentration after passage of the sample through the filterto that before passage of the sample through the filter was calculated,to thereby determine the filter-passing rate of the sample. The resultsare illustrated in Table 11.

TABLE 11 Concentration of cross-linked CS Viscosity Passing rate SampleCompound (wt %) (mPa · s) (%) Sample 36 Compound 10 2.00 52 99 Sample 22Compound 1 10.00 3000 96 Sample 26 Compound 6 7.49 4340 97 Sample 31Compound 7 6.00 8190 99 Sample 35 Compound 8 4.00 10649 95 Sample 30Compound 7 4.00 2880 >99 Sample 34 Compound 8 2.00 2800 89 Sample 55Compound 48 2.00 38 >99 Sample 56 Compound 49 2.00 60 >99 Sample 57Compound 50 2.00 122 98 Sample 58 Compound 51 2.00 27 >99 Sample 59Compound 52 2.00 62 98 Sample 60 Compound 53 2.00 130 97 Sample 61Compound 54 2.00 42 99 Sample 62 Compound 55 2.00 98 98 Sample 63Compound 56 2.00 45 >99 Sample 64 Compound 57 2.00 72 >99 Sample 65Compound 58 2.00 191 96 Sample 66 Compound 59 2.00 49 >99 Sample 67Compound 60 2.00 88 >99 Sample 68 Compound 61 2.00 44 95 Sample 69Compound 62 2.00 100 >99 Sample 70 Compound 63 2.00 50 96 Sample 71Compound 64 2.00 123 >99 Sample 75 Compound 65 2.00 51 >99 Sample 76Compound 66 2.00 157 99 Sample 77 Compound 67 2.00 85 >99

<Conclusion>

The results demonstrated that the samples prepared in Example 13 exhibita filter-passing rate of 80% or more.

Example 16

Examination for Effect of Cross-Linked CS in Accelerating Healing ofCorneal Epithelial Disorder (1)

SD rats (Charles River Laboratories Japan, Inc.) were prepared into dryeye models in accordance with the method described in Fujihara, et al.(Invest. Ophthalmol. Vis. Sci. 42 (1): 96-100 (2001)). A test substancewas instilled into the eyes of each model and examined for the effect ofaccelerating the healing of corneal epithelial disorder.

<Test Substance>

CS (CSC, sodium chondroitin sulfate (Japanese Pharmaceutical Codex),weight average molecular weight: 40,000, Seikagaku Corporation),compound 10, compound 18, or compound 42 was mixed with PBS so as toachieve a CS (or cross-linked CS) concentration of 2%, and the mixturewas shaken by means of a shaking apparatus overnight, to thereby preparea test substance. The test substance was 2% CS, 2% cross-linked CS—NC2N,2% cross-linked CS—NC4N, or 2% cross-linked CS-OrnEt. PBS was used as acontrol.

<Method>

(1) Preparation of Dry Eye Model

The exorbital lacrimal gland was excised from both eyes of an SD ratunder inhalation anesthesia with isoflurane, to thereby prepare a dryeye model.

(2) Ocular Instillation of Test Substance

About eight weeks after the preparation of the model, a portion ofcorneal epithelial disorder was stained with fluorescein. The degree ofstaining at an upper, middle, or lower portion of the superficial corneawas scored according to the following classification, and the totalscores of each portion were calculated. For an individual exhibiting abinocular average score of 5 or more, the test substance was instilledinto both eyes (5 μL per an eye) twice a day for three weeks. PBSserving as a control was instilled in the same manner as describedabove.

<Criteria for Determination>

Score 0: No dot stained

Score 1: Sparse (fluorescein staining dots are distant from each other)

Score 2: Moderate (intermediate between scores 1 and 3)

Score 3: Dense (most of fluorescein staining dots are close each other)

(3) Evaluation for Effect of Healing Corneal Epithelial Disorder

One, two, and three weeks after the initiation of ocular instillation,the effect of healing corneal epithelial disorder was scored accordingto the method described in (2) above.

(4) Statistical Analysis

One, two, and three weeks after the initiation of ocular instillation,the difference in averaged score between PBS and the test substance wasanalyzed by t-test with a significance level of 5% (two-sided).

<Test Results>

Table 12 and FIG. 4 illustrate the transitions and averages of scoresfor one, two, and three weeks. 2% Cross-linked CS—NC2N, 2% cross-linkedCS—NC4N, and 2% cross-linked CS-OrnEt exhibited a significant effect ofaccelerating the healing of corneal epithelial disorder as compared withPBS. 2% Cross-linked CS—NC2N and 2% cross-linked CS—NC4N exhibited asignificant effect of accelerating the healing of corneal epithelialdisorder as compared with 2% CS.

TABLE 12 PBS 2% CS 2% CS-NC2N 2% CS-NC4N 2% CS-OrnEt Before ocularinstillation 6.8 ± 0.5 6.2 ± 0.4 6.6 ± 0.5 6.4 ± 0.4 6.6 ± 0.4 1 weekafter 5.0 ± 0.7 5.1 ± 0.5 4.0 ± 0.9 4.8 ± 0.4 5.1 ± 0.7 2 weeks after5.6 ± 0.5 4.7 ± 0.5 3.1 ± 0.7 3.2 ± 0.4 4.0 ± 0.6 3 weeks after 5.5 ±0.5 5.2 ± 0.4 3.2 ± 0.5 3.6 ± 0.6 3.2 ± 0.4 n number n = 10 n = 10 n =10 n = 10 n = 10 Average ± standard error

Example 17

Examination for Effect of Cross-Linked CS in Accelerating Healing ofCorneal Epithelial Disorder (2)

The effect of accelerating the healing of corneal epithelial disorderwas examined in the same manner as described in Example 16.

<Test Substance>

Compound 49 or compound 52 was mixed with PBS so as to achieve across-linked CS concentration of 2%, and the mixture was shaken by meansof a shaking apparatus overnight, to thereby prepare a test substance.The test substance was 2% cross-linked CS-triAmine and 2% cross-linkedCS—NC3(OH)N. PBS was used as a control (Example 17A).

Compound 55 or compound 60 was mixed with PBS in the same manner asdescribed above, to thereby prepare a test substance. The test substancewas 2% cross-linked CS—NC4(═)N and 2% cross-linked CS-Cyclohex. PBS wasused as a control (Example 17B).

<Test Results>

Table 13 and FIG. 5 (Example 17A) and Table 14 and FIG. 6 (Example 17B)illustrate the transitions and averages of scores for one, two, andthree weeks. 2% Cross-linked CS-triAmine, 2% cross-linked CS—NC3(OH)N,2% cross-linked CS—NC4(═)N, and 2% cross-linked CS-Cyclohex exhibited asignificant effect of accelerating the healing of corneal epithelialdisorder as compared with PBS.

TABLE 13 2% 2% PBS CS-triAmine CS-NC3(OH)N Before ocular instillation7.0 ± 0.8 6.9 ± 0.6 7.4 ± 0.8 1 week after 6.0 ± 0.8 4.0 ± 0.6 6.3 ± 0.62 weeks after 5.4 ± 0.3 4.1 ± 0.8 3.8 ± 0.8 3 weeks after 5.2 ± 0.5 3.6± 0.8 2.5 ± 0.3 n number n = 10 n = 10 n = 10 Average ± standard error

TABLE 14 2% PBS 2% CS-NC4(═)N CS-Cyclohex Before ocular instillation 6.7± 0.5 6.7 ± 0.4 6.7 ± 0.5 1 week after 5.9 ± 0.6 4.0 ± 0.4 4.1 ± 0.3 2weeks after 5.7 ± 0.8 4.6 ± 0.8 4.1 ± 0.4 3 weeks after 6.4 ± 0.8 3.4 ±0.7 4.5 ± 0.6 n number n = 10 n = 10 n = 10 Average ± standard error

Example 18

Examination for Effect of Cross-Linked CS in Accelerating Healing ofCorneal Epithelial Disorder (3)

The effect of accelerating the healing of corneal epithelial disorderwas examined in the same manner as described in Example 16.

<Test Substance>

Compound 26, compound 62, compound 64, compound 66, or compound 67 wasmixed with PBS so as to achieve a cross-linked CS concentration of 2%,and the mixture was shaken by means of a shaking apparatus overnight, tothereby prepare a test substance. The test substance was 2% cross-linkedCS—NC6N, 2% cross-linked CS—NC5(S)N, 2% cross-linked CS-Glycol(C5), 2%cross-linked CS-Glycol(C11), and 2% cross-linked CS-Spermidine. PBS wasused as a control (Examples 18A to 18D).

<Test Results>

Table 15 and FIG. 7 (Example 18A), Table 16 and FIG. 8 (Example 18B),Table 17 and FIG. 9 (Example 18C), and Table 18 and FIG. 10 (Example18D) illustrate the transitions and averages of scores for one, two, andthree weeks. 2% Cross-linked CS—NC6N, 2% cross-linked CS—NC5(S)N, 2%cross-linked CS-Glycol(C5), 2% cross-linked CS-Glycol(C11), and 2%cross-linked CS-Spermidine exhibited a significant effect ofaccelerating the healing of corneal epithelial disorder as compared withPBS. The averages of scores for one, two, and three weeks of 2%cross-linked CS-Glycol(C11) were lower than those of 2% cross-linkedCS-Glycol(C5). The results demonstrated that a larger number of oxygenatoms in the main chain leads to a stronger effect of accelerating thehealing of corneal epithelial disorder.

TABLE 15 PBS 2% CS-NC6N Before ocular instillation 5.7 ± 0.3 5.8 ± 0.3 1week after 4.9 ± 0.4 3.8 ± 0.5 2 weeks after 5.2 ± 0.3 3.9 ± 0.4 3 weeksafter 5.1 ± 0.3 4.4 ± 0.3 n number n = 10 n = 10 Average ± standarderror

TABLE 16 2% CS- 2% CS- PBS Glycol(C5) Glycol(C11) Before ocularinstillation 5.8 ± 0.2 5.7 ± 0.2 5.3 ± 0.3 1 week after 5.6 ± 0.2 5.0 ±0.3 4.7 ± 0.4 2 weeks after 5.4 ± 0.2 4.8 ± 0.3 4.3 ± 0.4 3 weeks after5.6 ± 0.2 5.0 ± 0.2 4.3 ± 0.2 n number n = 10 n = 10 n = 6 Average ±standard error

TABLE 17 PBS 2% CS-NC5(S)N Before ocular instillation 6.1 ± 0.3 6.1 ±0.3 1 week after 5.5 ± 0.3 4.7 ± 0.3 2 weeks after 4.8 ± 0.2 4.0 ± 0.3 3weeks after 5.3 ± 0.4 4.1 ± 0.2 n number n = 10 n = 10 Average ±standard error

TABLE 18 PBS 2% CS-Spermidine Before ocular instillation 6.7 ± 0.2 6.8 ±0.3 1 week after 5.8 ± 0.2 5.5 ± 0.3 2 weeks after 6.4 ± 0.3 5.3 ± 0.3 3weeks after 6.0 ± 0.4 5.1 ± 0.4 n number n = 10 n = 10 Average ±standard error

<Conclusion>

As illustrated above, the cross-linked CS of the present invention canbe used as an agent for the treatment of an eye disease, in particular,an agent for the therapy of a corneal epithelial disorder and/or dryeye.

INDUSTRIAL APPLICABILITY

The cross-linked CS of the present invention or a composition containingthe same can be industrially used as an agent for the treatment of aneye disease. In addition, the method of the present invention can beindustrially used as a method for the treatment of an eye disease.

I claim:
 1. A method for producing a cross-linked chondroitin sulfate ora pharmaceutically acceptable salt thereof, the method comprising:reacting a chondroitin sulfate or pharmaceutically acceptable saltthereof with an amine having two or more primary amino groups in asolvent comprising a condensing agent to obtain the crosslinkedchondroitin sulfate or a pharmaceutically acceptable salt thereof,wherein a mole equivalent of the amine is from 0.005 to 0.250 relativeto 1.00 mole equivalent of disaccharide unit of the chondroitin sulfateor pharmaceutically acceptable salt thereof, and wherein the amine has abiodegradable moiety represented by the following formula (II) in a mainchain thereof:—C(═O)-D-  (II) wherein, in the formula (II), D represents an oxygenatom or a sulfur atom.
 2. The method according to claim 1, wherein apercent of cross-linking of the cross-linked chondroitin sulfate or thepharmaceutically acceptable salt thereof is from 0.01% to 30%.
 3. Themethod according to claim 2, wherein the percent of cross-linking of thecross-linked chondroitin sulfate or the pharmaceutically acceptable saltthereof is from 0.05% to 10%.
 4. The method according to claim 3,wherein the percent of cross-linking of the cross-linked chondroitinsulfate or the pharmaceutically acceptable salt thereof is from 0.1% to5%.
 5. The method according to claim 1, wherein the condensing agent is4-(4, 6-dimethoxy-1, 3, 5-triazin-2-yl)-4-methylmorpholinium chloride.6. The method according to claim 1, wherein a mole equivalent of thecondensing agent is from A, wherein A=0.05×valence of the amine, to B,wherein B=5.00×valence of the amine, relative to 1.00 mole equivalent ofthe amine.
 7. The method according to claim 1, wherein the solvent iswater or a mixture of water and a water-miscible organic solvent.
 8. Themethod according to claim 7, wherein the water-miscible organic solventis at least one selected from the group consisting of methanol, ethanol,isopropanol, n-propanol, tertiary butanol, ethylene glycol monomethylether, ethyleneglycol monoethyl ether, acetone, 1,4-dioxane,tetrahydrofuran and acetonitrile.
 9. The method according to claim 1,wherein the amine is a diamine.